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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 282:397–402, 1997
Vol. 282, No. 1
Printed in U.S.A.
MK-801 Limits Neurovascular Dysfunction during Experimental
Allergic Encephalomyelitis1
CHRISTOPHER BOLTON and CAROLYN PAUL
Pharmacology Group, School of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, United Kingdom
Accepted for publication March 24, 1997
Enhanced permeability of the blood-brain barrier (BBB) is
a characteristic abnormality observed during the pathogenesis of the human demyelinating disease, multiple sclerosis
(MS) (Adams et al., 1989). The constitutive endothelial cells
of the neurovasculature operate selectively to maintain BBB
homeostasis but malfunction during MS to allow the formation of vasogenic edema and infiltration of inflammatory cells
into parenchymal tissues with subsequent demyelination of
nerve fibers. The inducible autoimmune condition experimental allergic encephalomyelitis (EAE) has many pathological features in common with MS, of which cerebrovascular
leakage in susceptible animals is a standard occurrence (Leibowitz and Kennedy, 1972; Hawkins et al., 1990). Original
pharmacological studies by us in acute EAE showed that the
glucocorticoid dexamethasone and the immunosuppressant
cyclosporin A limit neurovascular breakdown, possibly
through a reduction in the release of permeability-inducing
factors (Paul and Bolton, 1994, 1995).
Although the mechanisms instigating loss of BBB integrity
Received for publication December 3, 1996.
1
The investigation was supported financially by The Multiple Sclerosis
Society of Great Britain and Northern Ireland.
disruption in medulla-pons (P , .05) and cervical spinal tissues
(P , .01). High-dose treatment also restricted disease development (P , .01) and lesion formation (P , .05). Therapeutic
MK-801, at 0.30 mg kg21 body weight, completely counteracted neuroendothelial leakage in cerebella (P , .05) and inhibited BBB dysfunction in remaining tissues without restricting
inflammatory cell invasion. However, doubling the dose did not
further enhance suppression of neurovascular breakdown. Our
use of MK-801 to control major features of EAE strongly implicates N-methyl-D-aspartate receptor-dependent mechanisms
in disease development and prompts consideration of a role for
the receptor in the pathogenesis of human demyelinating conditions.
in MS and EAE remain unclear, investigations in nonimmune models of neurovascular damage have identified central nervous system (CNS)-derived polyamines and the nitrogen intermediate nitric oxide (NO) as primary mediators of
cerebral vessel disruption (Koenig et al., 1983a; Trout et al.,
1986; Faraci and Brian, 1994). Interestingly, studies in EAE
and MS have indicated cerebral NO involvement (Koprowski
et al., 1993; Bö et al., 1994), and preliminary observations by
us have shown enhanced NO and polyamine levels in neurovascular isolates from EAE-diseased animals corresponding
with initial BBB leakage (Bolton et al., 1994; Scott et al.,
1996). Furthermore, dexamethasone and cyclosporin A have
the potential to inhibit CNS vessel polyamine and NO production (Koenig et al., 1989; Ryffel, 1993), which supports our
original hypothesis concerning the drugs’ direct effects on the
BBB during early EAE.
Both NO and the polyamines can be generated by activation of the N-methyl-D-aspartate (NMDA) receptor which has
been linked to the neuronal injury seen in several neurodegenerative conditions including Parkinson’s and Alzheimer’s
disease (Lee et al. 1988; Zeevalk et al., 1994). In addition to
the well-demonstrated neuronal location, the NMDA subtype
of glutamate receptor has been shown to reside in rat CNS-
ABBREVIATIONS: BBB, blood-brain barrier; bwt, body weight; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; EVBE,
extravascular blood equivalent; 125I, 125iodine; 111In, 111indium; MS, multiple sclerosis; NMDA, N-methyl-D-aspartate; NO, nitric oxide; PBS,
phosphate-buffered saline; PI, postinoculation; HEPES, N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid.
397
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ABSTRACT
Increased permeability of the blood-brain barrier (BBB) is a
characteristic of the demyelinating disease multiple sclerosis
and the animal counterpart experimental allergic encephalomyelitis (EAE). In physically traumatized cerebral tissue neurovascular damage, linked with activation of the cerebroendothelialbound N-methyl-D-aspartate receptor, can be treated with the
antagonist MK-801. We have examined the ability of MK-801 to
modify BBB leakage and the development of disease during
EAE. Prophylactic MK-801, at 0.15 mg kg21 body weight suppressed neurovascular breakdown, measured by a dual radioisotope technique, and significantly reduced neurological deficits (P , .05), but not perivascular lesions. A 2-fold increase in
administered MK-801 completely prevented abnormal extravasation in cerebella (P , .01) and significantly inhibited BBB
398
Bolton and Paul
Vol. 282
Methods
Animals
Male Lewis rats, weighing 200 to 250 g, were used from stock bred
on site and housed five rats per cage, with food (CRM diet) and water
ad libitum.
Induction of EAE
EAE was induced in animals as described previously (Bolton and
Flower, 1989). An emulsion comprising equal parts of guinea pig
spinal cord, sterile PBS and incomplete Freund’s adjuvant (Difco
Laboratories, Detroit, MI) was prepared and supplemented with 10
mg ml21 Mycobacterium tuberculosis H37Ra (Difco Laboratories).
Rats were inoculated with 0.1 ml of inoculum into each hind footpad.
A minimum of 5 rats were used per treatment.
Quantitation of BBB Integrity
BBB permeability in selected areas of the CNS was determined
according to our previous methods (Paul and Bolton, 1995), which
are briefly detailed below.
Labeling of red blood cells with 111In-tropolonate. Cell-free
plasma was prepared from pooled Wistar rat blood by repeated
centrifugation. The remaining blood cells were resuspended in
HEPES saline buffer (20 mM HEPES [Gibco, Ltd., Paisley, UK];
0.8% NaCl), washed repeatedly to remove leukocytes and reconstituted to provide an erythrocyte concentration of 5 3 108 cells ml21.
A preparation containing 20 mCi 111In-tropolonate/5 3 108 red blood
cells was incubated at 37°C for 20 min followed by washing and
resuspension at 5 3 109 cells/0.5 ml cell-free plasma.
Determination of BBB permeability. Rats received 10 mCi
125
I-rat serum albumin i.v. under halothane/oxygen and, 24 hr later,
5 3 109 111In-red blood cells were injected as a blood volume marker.
After a 4.5-min circulation time, cardiac blood was collected into
heparin-coated tubes followed by a lethal injection of euthatal (RMB
Animal Health Ltd., Dagenham, UK) at 5 min. Cerebella, medullapons and cervical spinal tissues were dissected out and the 111In
levels in samples and 100-ml blood aliquots from each animal were
recorded by an LKB minigamma counter. Quantities of 125I were
measured in samples following 111In decay after storage for 3 weeks
at 220°C. BBB permeability, expressed as EVBE, was calculated
from isotope levels in tissue and blood (equation 1) and is a measure
of radiolabeled albumin that has crossed the neurovasculature and
accumulated within CNS tissues.
125
111
125
111
I Tissue cpm/g
2
I Blood cpm/ml
In Tissue cpm/g
3 100 5 EVBE
In Blood cpm/ml
(1)
Corticosterone Radioimmunoassay
Animals were weighed daily beginning on day 0 PI and assessed
for neurological EAE. Disease symptoms appeared after weight loss
and were scored as follows: 1, flaccid tail; 2, hind limb hypotonia; 3,
partial hind limb paralysis; 4, complete hind limb paralysis.
Circulating corticosterone levels in rats were determined to exclude the possibility that treatment regimes enhanced endogenous
steroid levels which are known to influence the course of EAE
(MacPhee et al., 1989) and could therefore contribute to drug efficacy. Blood was collected at a standard time by cardiac puncture,
dispensed into heparin-coated tubes for subsequent extraction of
plasma by centrifugation at 300 3 g with storage at 220°C before
assay. Plasma corticosterone levels were measured in samples from
all treatment groups with a Gamma-B 125I-Corticosterone Radioimmunoassay Kit (IDS, Tyne and Wear, UK) according to the manufacturer’s instructions.
Assessment of Histological EAE
Statistical Analysis
The cervical spinal cords of animals from vehicle and drug treatment groups were examined by light microscopy for inflammatory
lesions sampling on day 12 PI. Spinal tissue was selected for analysis
because a heavy lesion load can be guaranteed in this CNS area
during the onset of acute EAE (Bolton et al., 1984). Furthermore, the
efficacy of MK-801 to restrict lesion development during early EAE
could be evaluated confidently. The upper 1.5 cm of tissue was
dissected and snap frozen. Cervical cord sections were cut at 5-mm
thickness, at one standard depth and stained with hematoxylin and
eosin. Lesion number per section was quantitated “blind” and assessed for intensity of cellular infiltration.
Plasma corticosterone data were analyzed by one-way analysis of
variance. Significant differences between all other drug and vehicle
treatment results were determined using the Mann-Whitney U test
for nonparametric data with Bonferroni correction for multiple comparisons where required.
Evaluation of Neurological EAE
Preparation and Administration of MK-801
MK-801 (supplied by Dr. L.L. Iversen, Merck, Sharp and Dohme
Research Laboratories, Harlow, England) was suspended in sterile
PBS and administered prophylactically by i.p. injection, once daily at
either 0.15 mg kg21 bwt or 0.3 mg kg21 bwt for 6 days, beginning day
7 PI. Animals were also treated therapeutically with MK-801 starting from initial weight loss, typically day 10 PI, for 3 days dosing i.p.,
once daily at either 0.3 mg kg21 bwt or 0.6 mg kg21 bwt. Control
EAE-inoculated rats received vehicle alone.
Results
The effects of prophylactic MK-801 administration
on neurovascular permeability and disease development. The appearance of neurological EAE in vehicletreated, sensitized rats, 12 days PI, was accompanied by
increased vascular permeability in all CNS areas studied and
was similar to previous results from untreated diseased animals detailed in our earlier studies (fig. 1) (Paul and Bolton,
1995). Repeated prophylactic doses of MK-801 at 0.15 mg
kg21 bwt suppressed BBB leakage and significantly reduced
neurological deficits in EAE-diseased rats (P , .05) (table 1).
However, a 2-fold increase in the administered dose of MK801 maintained EVBE values within normal limits in the
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derived blood vessels (Garthwaite et al., 1988; Monaghan et
al., 1989; Koenig et al., 1992), although species variation in
receptor expression at the neuroendothelium is recognized
(Beart et al., 1988; Faraci and Breese, 1993; Giese et al.,
1995). Up-regulation of the neuroendothelial NMDA receptor
and associated permeability-inducing factor release culminate in the loss of BBB integrity in nondisease models, which
can be suppressed by the specific noncompetitive antagonist
MK-801 (dizocilpine maleate) (Koenig et al., 1992). Interestingly, the potential for NMDA receptor activation exists in
EAE as increased levels of excitatory amino acid agonists
have been reported in the CNS of animals during disease
(Honegger et al., 1989; Flanagan et al., 1995). Therefore, we
were interested in examining the ability of MK-801 to suppress aberrant CNS vessel leakage during EAE and thereby
provide clear evidence for NMDA receptor involvement in
neurovascular breakdown in an immune-based model of human neurological disease.
1997
cerebella of treated rats (P , .01) and significantly inhibited
BBB disruption in medulla-pons (P , .05) and cervical spinal
tissue (P , .01). In addition, high-dose MK-801 markedly
curtailed the development of paralytic disease in EAE-inoculated animals (P , .01). Body weight loss in drug-treated
groups was not in excess of that shown by vehicle controls,
indicating the dosing regimes used were well tolerated. Prophylactically treated animals receiving 0.3 mg kg21 bwt MK801 lost significantly less weight, from disease onset, than
vehicle-dosed rats (P , .05) (table 1).
The effects of therapeutic MK-801 administration on
neurovascular permeability and disease development.
Vascular leakage in defined areas of the CNS from inoculated, vehicle-treated rats were similar to values recorded for
prophylactically dosed controls (fig. 2). Therapeutic doses of
MK-801 at 0.3 mg kg21 bwt completely counteracted neuroendothelial disruption in the cerebella of treated rats (P ,
.05) and markedly reduced BBB dysfunction in remaining
isolated tissues. However, increasing the dose of drug to 0.6
mg kg21 bwt did not further enhance suppression of neurovascular breakdown to a significant level compared with the
inhibition achieved with the lower-dose therapy. The mean
neurological scores for animals receiving either 0.3 mg kg21
bwt or 0.6 mg kg21 bwt were not significantly different from
vehicle controls (table 1).
Evaluation of CNS lesions after MK-801 treatment.
Prophylactic administration of MK-801 at 0.15 mg kg21 bwt
from day 7 PI did not reduce the number of inflammatory
lesions in cervical spinal tissues or limit the intensity of
infiltration (table 2). However, increasing the dosage significantly reduced the density of perivascular lesions (P , .05).
Furthermore, the extent of inflammatory cell infiltration was
markedly restricted compared with vehicle control tissues
(P , .02). Therapeutic treatments of 0.3 mg kg21 bwt and 0.6
mg kg21 bwt MK-801 did not alter the appearance or severity
of inflammatory infiltrates.
The effect of vehicle and MK-801 treatment regimes
on circulating corticosterone levels. Prophylactic vehicle
399
treatment resulted in elevated endogenous corticosterone
levels (table 1) similar to previous values recorded in untreated EAE-inoculated rats during the early onset of disease
(Mackenzie et al., 1989; Elderfield et al., 1993). Both prophylactic MK-801 treatments had circulating glucocorticoid levels above normal limits (32.9 6 20.6; n 5 5), but below vehicle
control values, this corresponding with the reduced severity
of symptoms.
Short-term therapeutic vehicle corticosterone values were
increased compared with normal levels but were less than
values recorded in samples from prophylactically treated
controls, although symptoms were similar. Systemic glucocorticoid concentrations in treatment groups were similar
to vehicle control results, which reflected the lack of improved disease severity.
The results confirm prophylactic and therapeutic administration of MK-801 does not induce an increase in circulating
corticosteroid levels above vehicle control values, which illustrates that drug effects are not mediated through the actions
of endogenous glucocorticoids.
Discussion
EVBE values recorded in target tissues from EAE-affected,
vehicle-dosed rats were equivalent to estimates previously
made by us in similar CNS isolates, reconfirming the occurrence of BBB breakdown during early disease and emphasizing the reproducibility of the radioisotope technique for quantitating neuroendothelial leakage (Paul and Bolton, 1995).
The present study demonstrates that prophylactic and therapeutic doses of the NMDA receptor antagonist and ion channel blocker MK-801 significantly suppresse neuroantigeninduced cerebrovascular disruption. The reduced symptom
expression observed after long-term administration of the
drug is in agreement with recent studies by Wallström et al.
(1996) using the glutamate receptor antagonist memantine
to inhibit the expression of EAE. However, although our
investigation clearly demonstrates limited perivascular lesions in the spinal cord 12 days PI after prophylactic treatment with high-dose MK-801, immunohistochemical assessment of cellular infiltration into the CNS suggested no
improvement after memantine treatment. Furthermore, our
studies confirm MK-801 effects on the course of EAE are not
mediated through excess circulating glucocorticoids, which
have previously been shown by us and others to influence
BBB permeability and determine the course of EAE (Bolton
and Flower, 1989; MacPhee et al., 1989; Paul and Bolton,
1995).
Increased permeability of cerebral vessels during the
course of MS or with the onset and development of EAE has
been well recognized and acknowledged as an intrinsic feature of both conditions (Barlow, 1956; Broman, 1964; Leibowitz and Kennedy, 1972; Gay and Esiri, 1991). However, and
despite subsequent studies describing physiological and biochemical abnormalities associated with BBB dysfunction
(Kristensson and Wisniewski, 1977; Claudio et al. 1989;
Hawkins et al., 1990, 1992), the events which precipitate
neuroendothelial leakage remain unclear. Pharmacological
studies by us and others have offered an additional approach
to elucidating the mechanisms which trigger abnormal leakage at CNS vascular sites (Claudio and Brosnan, 1992;
O’Neill et al., 1992; Bolton et al., 1994; Paul and Bolton,
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Fig. 1. The ability of prophylactically administered MK-801 to suppress neurovascular permeability in EAE-inoculated Lewis rats. MK801 treatment was initiated on day 7 PI and continued for 6 days dosing
i.p. at either 0.15 mg kg21 bwt (hatched columns, n 5 6) or 0.3 mg kg21
bwt (grey columns, n 5 6). Vehicle control EAE-sensitized animals (solid
column, n 5 10) received sterile PBS. Normal neurovascular permeability was demonstrated in uninoculated Lewis rats (open column, n 5
6). Columns represent mean EVBE values 6 S.E. * P , .05 and ** P ,
.01 compared with vehicle treatment.
MK-801 Limits Neurovascular Damage
400
Bolton and Paul
Vol. 282
TABLE 1
Effects of MK-801 administration on neurological status, weight loss and plasma corticosterone levels
Prophylactic
Vehicle
0.15 mg MK-801
0.30 mg MK-801
Therapeutic
Vehicle
0.30 mg MK-801
0.60 mg MK-801
n
Neurological
Score 6 S.E.a
Weight Loss 6
S.E. (g)b
Plasma Corticosterone
6 S.E. (ng/ml)
11
6
6
2.73 6 0.35
1.08 6 0.42*
0.50 6 0.41**
17.82 6 1.41
16.50 6 3.48
9.33 6 4.37*
104.5 6 21.0
48.5 6 12.2
67.5 6 8.12
2.80 6 0.64
2.25 6 0.75
1.00 6 0.43
15.80 6 2.40
15.33 6 2.35
19.33 6 2.27
53.6 6 5.28
72.0 6 16.0
66.3 6 15.6
5
6
6
a
Neurological scores observed at the end of the dosing period.
Weight loss during the final 3 days of treatment.
* P , .05 and ** P , .01, Mann-Whitney U test.
b
TABLE 2
Intensity and number of inflammatory lesions in the cervical
spinal cord after MK-801 treatment
Lesion Intensity
1/2
1
11
111
1111
Mean Lesion
Number
6 S.E.
0
0
4
4
2
1
2
1
0
2
3
1
2
0
0
69 6 10
61 6 15
27 6 19**
0
0
0
2
3
2
2
2
2
1
1
1
0
0
0
43 6 8
40 6 9
51 6 14
n
Prophylactic
Vehicle
10
0.15 mg MK-801
6
0.3 mg MK-801
6*
Therapeutic
Vehicle
5
0.3 mg MK-801
6
0.6 mg MK-801
5
Mann-Whitney U test analysis of data showed significant differences from
vehicle controls of * P , .02 for lesion severity and ** P , .05 for lesion number.
1995). In particular, the use of specific antagonists to characterize receptor-mediated events in BBB breakdown is exemplified through sequential investigations with the quinazoline derivative prazosin to identify the intimate
involvement of the cerebroendothelial alpha-1 adrenoceptor
in neurovascular dysfunction during EAE (Brosnan, 1985;
Brosnan et al., 1986; Goldmuntz et al., 1986; Claudio and
Brosnan, 1992).
The usefulness of drug-receptor studies to determine
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Fig. 2. The ability of therapeutically administered MK-801 to suppress
neurovascular permeability in EAE-inoculated Lewis rats. Treatment
with MK-801 at either 0.3 mg kg21 bwt (hatched columns, n 5 6) or 0.6
mg kg21 bwt (grey columns, n 5 5) commenced at weight loss and
continued for 3 days dosing i.p. once daily. Vehicle control EAEsensitized animals (solid column, n 5 6) received sterile PBS. Normal
neurovascular permeability was demonstrated in uninoculated Lewis
rats (open column, n 5 6). Columns represent mean EVBE values 6
S.E. * P , .05 compared with vehicle treatment.
mechanisms inducing BBB disruption is well illustrated by
the related work of Koenig et al. (1992) and the recent studies
of Miller et al. (1996) who used nonimmune-mediated models
of barrier leakage and specific antagonists to identify the
cerebrovascular-located NMDA receptor as pivotal in the
control of CNS vessel permeability. In particular, Koenig and
co-workers showed that neuroendothelial NMDA receptor
activation, after binding of the amino acid agonist glutamate,
could be prevented by the antagonistic actions of MK-801.
Moreover, the study demonstrated that in vivo treatment
with the drug inhibited polyamine synthesis which these
workers had previously shown to cause BBB breakdown via
cytotoxic mechanisms (Koenig et al., 1983a, b, 1989). Interestingly, increased levels of NMDA receptor ligands such as
glycine and quinolinic acid have been found in EAE CNS
tissue (Honegger et al., 1989; Flanagan et al., 1995) providing
the potential to enhance receptor function. Therefore, increased excitatory amino acid availability during EAE may
indirectly account for our findings of excess polyamines in
neurovascular isolates coinciding with enhanced permeability described in the current study and earlier investigations
(Bolton et al., 1994; Paul and Bolton, 1994). Finally, more
recent work by us has shown that elevated polyamine levels
in CNS tissues from EAE-sensitized rats can be markedly
reduced by MK-801 treatment reinforcing a role for the compounds in BBB disruption (Paul et al., 1996).
An additional consequence of NMDA receptor activation is
the generation, via a constitutive synthase enzyme, of the
vasoactive nitrogen intermediate, NO (Garthwaite et al.,
1988, 1989; Southam et al., 1991). NMDA receptor-dependent NO production at cerebroendothelial sites has been
closely associated with changes in local blood flow and vascular tone, which could ultimately affect BBB permeability
(Faraci and Breese, 1993). In addition, more recent studies
have proposed that NO, produced by target tissues during
the onset and progression of EAE (Lin et al., 1993; Bolton et
al., 1994; Scott et al., 1994; Hooper et al., 1995), may give rise
to the generation of longer acting peroxynitrite products with
increased cytotoxic potential (Hooper et al., 1995). Indeed,
pharmacological studies by us, using selective inhibitors of
NO synthase enzymes, strongly support a secondary role for
the molecule in the pathogenesis of EAE (Scott et al., 1995;
1996). Therefore, MK-801 may correct abnormal BBB permeability by acting at neurovascular sites to limit NO generation and polyamine production by down-regulating vessel
NMDA receptor activation after stimulation by excess glutamate.
1997
Acknowledgments
The authors acknowledge the expert technical assistance of Mrs.
Lesley Moore and are grateful to the staff of the Cellular Pathology
Department at the Royal United Hospital for histologically processing tissues. We also thank Drs. L.L. Iversen, R.G. Hill and R.J.
Hargreaves of Merck, Sharp and Dohme Research Laboratories,
Harlow, UK, for useful discussions while conducting the studies.
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In addition to an action on neuroendothelial-associated
NMDA receptors MK-801 has well-documented effects on
receptors at central neuronal sites (Wong et al., 1986). Hence,
the drug could act indirectly to prevent BBB leakage by
inhibiting the production of neuronal-derived, permeabilityinducing factors disruptive at cerebroendothelial locations. A
recent study by Purcell et al. (1996) also provides evidence,
through the use of MK-801, for the presence of NMDA receptors on mast cells which appear to be involved in the pathogenesis of EAE (Bö et al., 1991; Levi-Schaffer et al., 1991).
Finally, the possibility that MK-801 may have, as yet, unknown peripheral effects which influence the development of
EAE cannot be ignored. However, we have excluded the
possibility that MK-801 is acting via the consequence of
up-regulated endogenous glucocorticoids and preliminary
data indicate the drug has no effects on in vitro lymphocyte
proliferation or macrophage function.
In conclusion, we have described the potent suppressive
actions of the NMDA receptor antagonist, MK-801, on BBB
breakdown, lesion formation and symptom onset during
early neurological EAE and thus identified a novel mechanism through which neurovascular disruption may occur.
NMDA receptor involvement in the pathogenesis of MS has,
to our knowledge, not been documented but clearly requires
investigation. Studies are ongoing to determine the precise
mode of action through which MK-801 corrects abnormal
BBB leakage during EAE and thereby indicate possible targets for the therapeutic control of MS.
MK-801 Limits Neurovascular Damage
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Bolton and Paul
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Send reprint requests to: Dr. C. Bolton, Pharmacology Group, School of
Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, UK,
BA2 7AY.
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