artigo sistema nervoso Guillain Barre

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Lovastatin attenuates nerve injury in an animal model of
Guillain–Barré syndrome
Jason P. Sarkey,*,� Michael P. Richards* and Evan B. Stubbs Jr�,§
*Research Service, Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines, Illinois, USA
�Program in Neuroscience, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA
�Neurology Service, Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines, Illinois, USA
§Departments of Neurology, Cell Biology, Neurobiology, and Anatomy, Stritch School of Medicine, Loyola University Chicago,
Maywood, Illinois, USA
Abstract
Statins, widely used as clinically effective inhibitors of
3-hydroxy-3-methylglutaryl coenzyme A reductase, exhibit
anti-inflammatory properties that may be of therapeutic
benefit for the management of some neurological disorders.
In this study, a short-term course of lovastatin treatment is
shown to markedly inhibit the development of experimental
autoimmune neuritis (EAN) in the absence of hepatotoxic or
myotoxic complications. Independent of cholesterol reduc-
tion, lovastatin treatment prevented EAN-induced peripheral
nerve conduction deficits and morphologic nerve injury.
Co-administration with mevalonate neutralized the prophy-
lactic effects of lovastatin. When administered therapeutic-
ally, lovastatin significantly shortened the disease course.
Autoreactive immunity, measured in vitro by myelin-stimu-
lated proliferation of splenocytes, was significantly dimin-
ished by in vivo lovastatin treatment. Th1-dominant immune
responses, measured by cytokine profiling, however, were
not affected by lovastatin. Sciatic nerves of lovastatin-trea-
ted immunized rats showed markedly reduced levels of
cellular infiltrates. Treating peripheral nerve endothelial
monolayers with lovastatin significantly inhibited the in vitro
migration of autoreactive splenocytes. Together, these data
demonstrate that a short-term course of lovastatin attenu-
ates the development and progression of EAN in Lewis rats
by limiting the proliferation and migration of autoreactive
leukocytes.
Keywords: experimental autoimmune neuritis, Guillain–
Barré syndrome, peripheral nerve, statins.
J. Neurochem. (2007) 100, 1265–1277.
Immunization of susceptible strains of laboratory animals
with an emulsion of bovine peripheral nerve myelin and
adjuvant induces an experimental autoimmune neuritis
(EAN) that closely models the pathogenicity of acute-onset
inflammatory demyelinating polyradiculopathy (AIDP)
(Waksman and Adams 1955). AIDP is a common North
American and European subgroup of the human PNS
disorder Guillain–Barré syndrome (GBS) (Kuwabara 2004).
In humans, the AIDP subgroup of GBS is hallmarked by a
rapid-onset monophasic course with severe ascending paresis
requiring, in some patients, mechanical ventilation. En-
hanced infiltration of inflammatory cells into peripheral
nerves (Kadlubowski et al. 1980; Prineas 1994; Kiefer et al.
2001; Maurer et al. 2002b) of affected patients is strongly
suggestive of an immune-mediated pathogenic process.
Cellular immunity directed against specific constituents of
the peripheral nerve myelin sheath is considered causal,
leading to debilitating segmental demyelination and, in some
cases, axonal loss (Hartung et al. 1988, 2001). In other cases,
affected patients develop a severe axonal subgroup of GBS
(an acute motor axonal neuropathy) that is distinct from
Received May 1, 2006; revised manuscript received August 22, 2006;
accepted August 22, 2006.
Address correspondence and reprint requests to Evan B. Stubbs Jr,
Research Service (151), Edward Hines Jr. VA Hospital, Hines, IL 60141,
USA. E-mail: evan.stubbs@med.va.gov
Abbreviations used: AIDP, acute-onset inflammatory demyelinating
polyradiculopathy; ALT, alanine transaminase; APC, antigen-presenting
cell; AST, aspartate transaminase; CFA, complete Freund’s adjuvant;
CK, creatine kinase; CMAP, compound muscle action potential; DMSO,
dimethylsulfoxide; EAE, experimental autoimmune encephalomyelitis;
EAN, experimental autoimmune neuritis; GBS, Guillain–Barré syn-
drome; HDL, high-density lipoprotein; HMG-CoA, 3-hydroxy-3-meth-
ylglutaryl coenzyme A; IL, interleukin; INF-c, interferon-c; LDL,
low-density lipoprotein; MHC, major histocompatibility complex; NCV,
nerve conduction velocity; p.i., post-immunization; PNVE, peripheral
nerve vascular endothelial; TNF-a, tumor necrosis factor-a.
Journal of Neurochemistry, 2007, 100, 1265–1277 doi:10.1111/j.1471-4159.2006.04309.x
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works 1265
AIDP. Clinical case studies have suggested that, in these
patients, the initiation of an autoimmune response occurs as a
result of an antecedent upper respiratory or gastroenteric
illness. An autoimmune etiology involving molecular mim-
icry is supported by studies describing serum antibody cross-
reactivity with pathogens isolated from affected acute motor
axonal neuropathy patients (Levin et al. 1998; Quarles and
Weiss 1999; Kuwabara et al. 2004; Ariga and Yu 2005). The
etiology of AIDP, however, is less understood and remains to
be the focus of intense investigation.
Insights into the immunological mechanisms responsible
for peripheral nerve demyelination or axonal injury in GBS
continue to emerge from studies of EAN. Active induction of
EAN with a variety of purified myelin proteins (Maurer et al.
2002a) or passive induction through the introduction of
myelin protein-specific autoreactive T cells (Linington et al.
1984) lends support to the thesis that early immune events in
GBS involve inappropriate recognition of myelin-expressed
self-antigens. Transendothelial migration of activated
lymphocytes into peripheral nerves of immunized animals
is an early key pathologic event, resulting in focal disruption
of the blood–nerve barrier (Spies et al. 1995; Hahn 1996).
Circulating autoreactive leukocytes may home to the PNS by
a mechanism that involves changes in nerve chemokine
expression (Zou et al. 1999; Kieseier et al. 2000). Upon
entering the PNS, it is thought that autoreactive leukocytes
encounter antigen-presenting cells (APCs) that display
appropriate neuritogenic cell-surface epitopes in association
with major histocompatibility complex (MHC) class II
antigens. Such an encounter would then result in T-lympho-
cyte proliferation and pro-inflammatory cytokine production,
thereby initiating a cascade of inflammatory responses
leading to nerve injury (Hartung et al. 1995).
Statins, including lovastatin, are a group of potent 3-
hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reduc-
tase inhibitors that are clinically approved for cholesterol
reduction. Increasing clinical and experimental evidence,
however, strongly supports anti-inflammatory effects of
statins. C-reactive protein, a marker of inflammation, is
reduced by statin treatment (Albert et al. 2001; Ridker et al.
2001). The effect of statins on the immune system is now
known to be pleiotropic, disrupting a number of key immune
signaling events including T-cell activation, proliferation,
and leukocyte trafficking (Danesh et al. 2003; Greenwood
et al. 2003). Recently, statins have been proposed to shift the
T-cell cytokine response from a pro-inflammatory Th1 to an
anti-inflammatory Th2 profile (Youssef et al. 2002; Aktas
et al. 2003). However, this effect of statins does not appear to
be universal (Gegg et al. 2005). These studies raise the
question as to whether statin therapy may be of general use in
the management of inflammatory diseases, including those
affecting the PNS.
In this study, we evaluated lovastatin for the management
of EAN, an inflammatory animal model of acute demyeli-
nating peripheral neuropathy (Sarkey et al. 2005). We report
that a short-term course of high-dose lovastatin can prevent-
atively and therapeutically attenuate the development of
EAN in Lewis rats by limiting the proliferation and migration
of autoreactiveleukocytes into affected peripheral nerves.
Although the clinical significance of this finding remains to
be evaluated, we postulate that short-term application of
lovastatin may be of clinical use in the management of
inflammatory peripheral nerve diseases, including some
clinical subgroups of GBS.
Materials and methods
Induction and clinical evaluation of active EAN
This study was conducted using protocols approved by the
Institutional Animal Care and Use Committee in accordance with
the principles of laboratory animal care (NIH Publication No. 86-23,
1985). All animals were housed in pairs and allowed to have
standard rat chow and water ad libitum and maintained on a 10 h/
14 h light/dark cycle. Adult male Lewis rats (initial body weight
300 g; Harlan Sprague–Dawley, Indianapolis, IN, USA) were
anesthetized with ketamine (100 mg/kg)–xylazine (5 mg/kg) and
actively immunized by injection into the right hind footpad with
100 lL of a freshly prepared fine-particle emulsion (1 : 1 v/v)
containing lyophilized bovine peripheral nerve myelin (3.0 mg dry
weight) suspended in sterile saline and incomplete Freund’s adjuvant
(Sigma-Aldrich, St Louis, MO, USA) supplemented with 10 mg/mL
heat-inactivated Mycobacterium tuberculosis (strain H37RA; Difco
Laboratories, Detroit, MI, USA). Adjuvant control rats were injected
in the same way except bovine peripheral nerve myelin was omitted.
Bovine peripheral nerves (cauda equina) were harvested from killed
animals (<2 h post-mortem) and stored at )80�C until used. Myelin
was extracted and purified by discontinuous sucrose gradient
centrifugation according to the method of Wiggins et al. (1975).
The washed myelin pellet was re-suspended in ice-cold water,
lyophilized, and stored at )80�C until used.
Rats were scored daily for EAN in a blinded manner by two
independent investigators. The severity of clinical signs was scored
as follows: 0 = no illness; 1 = flaccid tail; 2 = abnormal gait;
3 = moderate paraparesis; 4 = severe paraparesis; 5 = paraplegia.
Intermediate clinical signs were scored in increments of 0.5.
Immunization with 3 mg of purified bovine peripheral nerve myelin
reproducibly induced severe paraparesis that rarely progressed to
paraplegia. Rats with EAN exhibited a monophasic course with
disease onset, defined as a minimal score of 0.5, beginning
14 ± 0.3 days post-immunization (p.i.) (n = 13).
Administration of lovastatin
To determine whether statins can protect against the development of
EAN, daily doses of lovastatin [10 mg/kg body weight dissolved in
50% dimethylsulfoxide (DMSO)] were administered to immunized
rats by i.p. injection beginning on day 8 p.i. just prior to the onset of
clinical disease (Fig. 1a). Control EAN rats received daily injections
of vehicle (50% DMSO, 0.1 mL). The therapeutic potential of statins
was assessed by administering lovastatin (10 or 25 mg/kg body
weight in 50% DMSO, 0.2 mL) to immunized rats beginning on day
1266 J. P. Sarkey et al.
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
14 p.i. after clinical disease had begun (Fig. 1b). The dose and
parenteral route was chosen in accordance with previous studies on
the effects of lovastatin in related autoimmune diseases (Greenwood
et al. 2003; Gegg et al. 2005) and assuming species differences in
statin metabolism (Black et al. 1998; Stern et al. 2000).
Peripheral nerve conduction studies
Evoked compound muscle action potential (CMAP) amplitudes and
latencies of peripheral nerves were quantitated as previously
described (Lawlor et al. 2002). Briefly, sciatic nerves of anesthe-
tized rats were stimulated at the sciatic notch or at the ankle (tibial
nerve) with unipolar pin electrodes using supramaximal stimuli
(25 mA, 0.05-ms duration) at high frequency (1 Hz) for the direct
measurement of motor nerve (M-wave) responses. Unipolar pin
recording electrodes were used to record the evoked potentials from
the plantar muscles of the un-injected left hind foot. Individual
responses were amplified and recorded using a TECA Synergy
electromyograph system. For each animal, the M-wave was
measured as an average of 25 individual evoked responses, repeated
in triplicate. Motor nerve conduction velocity (NCV) was calculated
as the distance from notch-to-ankle divided by the difference of the
M-wave latency (notch–ankle). A heating pad was used to maintain
the body temperature of the rat at 37�C.
Neuropathology
At peak of disease following nerve conduction studies, sciatic nerves
were rapidly harvested and immediately fixed in ice-cold 2% p-
formaldehyde–2.5% glutaraldehyde phosphate-buffered solution for
24 h at 4�C. Nerves were post-fixed in 1% osmium tetroxide,
dehydrated, and embedded in Embed-812 (Electron Microscopy
Sciences, Fort Washington, PA, USA) (Feirabend et al. 1998). Serial
transverse sections (0.5 lm) were prepared with a Reichert Ultracut S
microtome (W. Nuhsbaum Inc., McHenry, IL, USA) and osmicated
sections were stained with toluidine blue. Stained sections were
viewed using a Leitz DMRB (W. Nuhsbaum Inc.) inverted phase-
contrast microscope equipped with a Leica Wild MPS photographic
system (W. Nuhsbaum Inc.). Serial transverse ultrathin sections
(80 nm)were stained with 5% uranyl acetate and counter-stainedwith
Reynolds lead citrate. Electron micrographs were prepared using a
Hitachi H-600 75-kV electron microscope (Hitachi, Tokyo, Japan).
Histopathological changes (demyelination, axonal damage, cellular
infiltration, and edema) in sciatic nerves were semi-quantitated from
light micrographs in a blinded manner by two independent investi-
gators using a four-point scoring system as follows: 0 = normal;
1 = mild demyelination, axonal damage, or cellular infiltrates;
2 = moderate demyelination, axonal damage, or cellular infiltrates;
3 = severe demyelination, axonal damage, or cellular infiltrates.
Infiltration of leukocytes into sciatic nerve of vehicle-treated or
lovastatin-treated bovine peripheral nerve myelin (BPNM)-immun-
ized rats was determined by quantitative immunohistochemical
analysis. At peak of disease, rats were killed and sciatic nerves
quickly removed and snap frozen in liquid nitrogen. Serial
longitudinal sections (7 lm) of frozen nerves were prepared with
a Leica CM 1850 Cryostat (W. Nuhsbaum Inc.) and stored at
)20�C until use. Nerve sections were thawed, fixed in ice-cold
acetone, and immersed in 0.3% H2O2 · 30 min to suppress
endogenous peroxidase activity. Treated sections were incubated
overnight at 4�C in the presence of a 1 : 50 dilution of mouse anti-
rat CD68 or anti-rat CD8 primary antibody (Serotec, Raleigh, NC,
USA) or incubated for 1 h at 23�C in the presence of a 1 : 1000
dilution of mouse anti-rat CD4 primary antibody (Serotec).
Immunostained sections were subsequently incubated for 30 min
at 23�C with a 1 : 200 (for CD4 or CD8) or a 1 : 400 (for CD68)
dilution of a biotinylated anti-mouse IgG secondary antibody.
Immunostained leukocyte infiltrates were visualized with a
commercially available ABC kit (Vector Laboratories, Inc.,
Burlingame, CA, USA). Infiltrating CD68+ macrophages and
CD4+ and CD8+ lymphocytes were quantitated in a blinded
manner by a single investigator, by counting five randomly chosen
0.5 mm2 fields per section at 200· magnification.
Proliferation and cytokine assay
Splenocytes were prepared from vehicle- or lovastatin-treated
immunized rats at peak of disease and cultured in RPMI-1640 media
* 
* * * 
* 
* 
* 
0 5 10 15 20 25 30 35
0 
1 
2 
3 
4 
(a) 
(b) 
vehicle 
Lov (10 mg/kg) 
Lov (10 mg/kg) + Mev (20 mg/kg) 
CFA control 
Days post-immunization
0 5 10 15 20 25 30 35 
Days post-immunization
C
lin
ic
al
 s
co
re
0 
1 
2 
3 
4 
C
lin
ic
al
 s
co
re
Lovastatin treatment begins 
Lovastatin treatment begins EAN + vehicle 
EAN + Lov (25 mg/kg) 
* 
* 
* 
* * * * * 
Fig. 1 Lovastatin attenuates the developmentof experimental auto-
immune neuritis (EAN). Clinical development of EAN in rats immun-
ized at day 0 with complete Freund’s adjuvant (CFA control, n = 8) or
CFA containing 3-mg purified bovine peripheral nerve myelin. (a) Prior
to or (b) after disease onset, immunized rats were treated (arrow) daily
for 14 or 7 days, respectively, with 50% dimethylsulfoxide (vehicle,
n = 13), lovastatin (Lov, n = 10), or lovastatin plus mevalonate
(Lov + Mev, n = 5). Data shown are the mean ± SEM clinical scores.
*p < 0.01 determined for days 15–21 (lovastatin vs. vehicle control);
Mann–Whitney non-parametric U-test analysis.
Lovastatin attenuates experimental autoimmune neuritis 1267
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/
L glutamine, 0.05 lmol/L 2-mercaptoethanol, 100 U/mL penicillin,
and 100 lg/mL streptomycin at a density of 1.0 · 106 cells/mL in the
presence or absence of bovine peripheral nerve myelin (0.0, 1.0, 10.0,
or 100 lg/mL) for 96 h. For the final 24 h of antigen stimulation,
0.5 lCi of [3H]thymidine (6.7 Ci/mmol) was added to each well.
Labeled cells were harvested and [3H]thymidine incorporation into
DNA determined using a Hewlett-Packard Model 2500 TR liquid
scintillation analyzer (Technical Alternatives, Inc., Ann Arbor, MI,
USA). Cell culture supernatants from parallel non-radiolabeled assays
were collected at 96 h for production of interferon-c (IFN-c), tumor
necrosis factor-a (TNF-a), interleukin (IL)-5, IL-10, and IL-12.
Cytokine concentrations in supernatant were quantitated simulta-
neously on a Bio-plex� system (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) using a rat cytokine/chemokine lincoplex kit
(LINCO Research, St Charles, MO, USA).
Flow cytometric analysis
Splenocytes (1.0 · 106 in phosphate-buffered saline containing 1%
fetal calf serum and 0.09% sodium azide) prepared from vehicle- or
lovastatin-treated immunized rats at peak of disease were incubated
for 30 min at 4�C in the presence of 1 lg FITC-conjugated mouse
anti-rat CD11b/c (clone OX-42) antibody and with 1 lg of
phycoerythrin (PE)-conjugated mouse anti-rat MHC class II (clone
OX-6), CD80 (clone 3H5), or CD86 (clone 24F) antibody (BD
Pharmingen, San Diego, CA, USA). Co-immunostained cells were
washed twice with ice-cold phosphate-buffered saline and re-
suspended in 100 lL of ice-cold-buffered 4% p-formaldehyde. After
20 min, fixed cells were washed twice and data were collected with
a FACSCanto (BD Biosciences, San Jose, CA, USA) and analyzed
with FlowJo software (Tree Star, Inc., Ahsland, OR, USA).
Measurements of cholesterol, high-density lipoprotein and
low-density lipoprotein cholesterol, triglycerides, alanine
transaminase, aspartate transaminase, and creatine kinase
The content of total cholesterol, cholesterol in high-density
lipoproteins (HDL-cholesterol), total triglycerides, and enzymatic
activities of alanine transaminase (ALT), aspartate transaminase
(AST), and creatine kinase (CK) in rat sera were quantitated by the
clinical chemistry laboratory at Edward Hines Jr. VA hospital using
automated colorimetric assays on a Dimension� clinical chemistry
system (Dade Behring Inc., Newark, DE, USA). The content of
cholesterol in low-density lipoproteins is reported as a calculated
difference from total cholesterol as follows: {(total choles-
terol) ) [HDL-cholesterol + (total triglycerides/5)]}.
Splenocyte migration assay
Primary cultures of Lewis rat peripheral nerve vascular endothelial
(PNVE) cells were prepared according to the method of Argall et al.
(1994). PNVE cells were grown to confluency on polycarbonate
Transwell� inserts (pore size of 8.0 lm; Corning Inc., Corning, NY,
USA). Confluent PNVE cell cultures were then incubated with
vehicle (10% ethanol) or lovastatin (1 lmol/L) for 24 h. Parallel
cultures of splenocytes, prepared separately from immunized rats,
were cultured (5.0 · 106 cells/mL) for 24 h in the presence of
10 lg/mL bovine peripheral nerve myelin. Following the addition of
macrophage inflammatory protein (MIP)-1a (100 ng/mL) to the
bottom well, autoreactive splenocytes (2.0 · 106 cells) were then
added to each PNVE cell culture insert. After 3 h at 37�C, the
number of splenocytes that migrated across the PNVE cell
monolayer was quantitated.
Statistical analysis
Data are expressed as the mean ± SEM of n observations unless
noted otherwise. Statistical significance of parametric data was
determined by Student’s t-test. Significance between multiple
experimental groups was determined by one-way ANOVA with a
Bonferroni or Dunnett’s multiple comparison post hoc analysis. For
statistical evaluation of clinical and neuropathological data, a Mann–
Whitney non-parametric U-test analysis was performed. In each
case, p < 0.05 was considered statistically significant.
Results
Induction of EAN in Lewis rats
Male Lewis rats injected with an emulsion of bovine
peripheral nerve myelin (3 mg dry weight, BPNM) and
complete Freund’s adjuvant (CFA) developed a reproducible
monophasic course of muscle weakness that initially presen-
ted as a loss of tail tone beginning 14 ± 0.3 days p.i. and
rapidly progressed to severe paraparesis by 19 ± 0.3 days. At
day 22 p.i., the first signs of clinical improvement became
apparent. By 27 days p.i., muscle weakness had resolved
without evidence of relapse (Fig. 1a). Coincident with disease
onset, immunized rats experienced a transient reduction in
body weight with a maximal loss seen at peak of disease
(86.1 ± 1.8% of day 10 weight). By comparison, rats injected
with an emulsion of sterile saline and CFA (1 : 1 v/v) in the
absence of BPNM exhibited a steady increase in body weight
(data not shown) without developing clinical signs of disease
(adjuvant control, Fig. 1a). Inoculation with emulsified CFA
alone was not without effect, however, and produced notable
swelling of the injected footpad. The local inflammatory
response produced by injecting adjuvant alone was not
unexpected and is consistent with reports by early investiga-
tors (Pearson 1956; Waksman et al. 1960) of adjuvant-
induced arthritis in Lewis rats receiving a single inoculum of
mycobacteria emulsified in oil (i.e. CFA). These data largely
confirm early reports of peripheral nerve antigen-dependent
induction of EAN in Lewis rats (Hahn et al. 1988; Hahn
1996; Kafri et al. 2002) and support the use of EAN as a
reliable model in which to evaluate the effectiveness of statins
as novel therapeutic agents for the treatment of inflammatory
demyelinating peripheral neuropathy.
Prophylactic and therapeutic potential of lovastatin
To determine whether statins can protect against the devel-
opment of EAN, lovastatin (10 mg/kg body weight) was
administered by i.p. injection beginning on day 8 p.i. just
prior to the onset of clinical disease. We found that daily
doses of lovastatin significantly attenuated the development
1268 J. P. Sarkey et al.
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
of EAN when compared with vehicle-treated controls
(Fig. 1a). Discontinuing lovastatin-treatment did not elicit a
relapse of EAN, indicating that lovastatin suppressed, rather
than delayed, disease development. To test whether lovast-
atin has therapeutic value for the treatment of EAN,
immunized rats were treated with lovastatin beginning on
day 14 p.i. after the onset of clinical disease. Therapeutic
administration of lovastatin at a prophylactic dose of 10 mg/
kg body weight did not, however, alter the development or
progression of EAN (data not shown). In Fig. 1b, we show
that increasing the dose of lovastatin to 25 mg/kg body
weight modestly attenuated the peak clinical severity of EAN
and significantly shortened the disease course when com-
pared with vehicle-treated controls (19.6 ± 0.8 days vs.
27.6 ± 1.2 days; p < 0.001). Discontinuinglovastatin ther-
apy did not elicit a relapse of EAN in affected rats (Fig. 1b).
Lovastatin protects against EAN-induced peripheral
nerve injury
The ability of lovastatin to protect against EAN-induced
peripheral nerve injury was quantitated in vivo by evoked-
response electrophysiology. Marked deficits in both NCVand
evoked CMAP amplitude were seen in vehicle-treated
immunized rats (Fig. 2). At peak of disease, sciatic NCV
was significantly slowed compared with velocities measured
prior to immunization or velocities from adjuvant control rats
(Fig. 2b). Evoked CMAP amplitudes were similarly reduced
in vehicle-treated immunized rats (Fig. 2c). In agreement
with the clinical findings shown in Fig. 1, prophylactic
lovastatin treatment prevented the development of these
peripheral nerve deficits (Fig. 2).
Representative light and electron micrographs of sciatic
nerves harvested at peak of disease from vehicle- or
lovastatin-treated immunized rats are shown in Fig. 3. Mixed
morphologic changes including evidence of inflammation
(cellular infiltration and edema) and of nerve injury (demy-
elination and axonal damage) were observed. A semi-
quantitative assessment of toluidine blue-stained transverse
sections (0.5 lm) demonstrated significant (p < 0.0001,
n = 5) histopathologic findings in sciatic nerves harvested
from vehicle-treated immunized rats (histopathological
score = 2.30 ± 0.31) compared with nerves harvested from
adjuvant controls (0.14 ± 0.17; micrographs not shown). In
contrast, sciatic nerves harvested from lovastatin-treated
immunized rats exhibited only sparse histopathologic find-
ings (0.18 ± 0.18, n = 5) that were statistically indistin-
guishable (p = 0.73) from adjuvant controls.
Vehicle control
Baseline Peak EAN 
Lovastatin 
Baseline Peak EAN 
Sciatic notch
(a) 
(b) 
(c) 
Ankle 
Sciatic notch
Ankle 
5 mV 
1 ms
5 mV 
1 ms
0
10
20
30
40
50
60
70
* 
EAN + Lovastatin EAN + Vehicle CFA control 
EAN + Lovastatin EAN + Vehicle CFA control 
baseline 
peak EAN 
sciatic notch 
ankle 
C
on
du
ct
io
n 
ve
lo
ci
ty
 (
m
/s
) 
0
20
40
60
80
100
120
** 
* 
C
M
A
P
 a
m
pl
it
ud
e 
(%
 o
f 
ba
se
lin
e)Fig. 2 Lovastatin protects against experimental autoimmune neuritis
(EAN)-induced peripheral nerve conduction deficits. (a) Representative
tracings of motor nerve compound muscle action potential (CMAP)
amplitudes evoked from the sciatic notch or ankle of rats and recorded
prior to (baseline) and 21 days (peak EAN) after immunization. Rats
were treated with 50% dimethylsulfoxide (vehicle control) or lovastatin
(10 mg/kg) prior to disease onset as indicated in Fig. 1a. (b, c)
Quantitative comparison of evoked motor nerve conduction velocities
(b) or of evoked CMAP amplitudes (c) recorded prior to (baseline, open
bars) and at peak of disease (shaded bars). Data shown are the
means ± SEM from four to eight rats per treatment group. **p < 0.01;
*p < 0.05, relative to complete Freund’s adjuvant (CFA) controls;
one-way ANOVA with Bonferroni multiple comparison post-test.
Lovastatin attenuates experimental autoimmune neuritis 1269
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
(a) (d)
(b) (e)
(c) (f)
Fig. 3 The effect of lovastatin on experi-
mental autoimmune neuritis-induced chan-
ges in peripheral nerve morphology.
Histology (a–c) and electron micrographs
(d–f) of representative transverse sections
of sciatic nerves harvested from immunized
rats treated prior to disease onset with (a, d)
50% dimethylsulfoxide vehicle control, (b,
e) lovastatin (10 mg/kg), or (c, f) lovastatin
(10 mg/kg) plus mevalonate (20 mg/kg).
Experimental autoimmune neuritis-induced
histopathological changes in nerve mor-
phology are indicated (arrows) for compar-
ison. Scale bar: 50 lm (a–c) and 3 lm
(d–f).
Table 1 Lipid content in sera of Lewis rats
Treatment group n
Total
cholesterol
HDL
cholesterol
LDL
cholesterol Triglyceride
Naı̈ve control 6 94.7 ± 1.4 28.2 ± 1.0 10.7 ± 0.2 110.8 ± 18.1
Adjuvant control 5 91.2 ± 3.4 24.8 ± 1.4 9.8 ± 0.6 91.6 ± 6.2
Vehicle-treated EAN 5 67.8 ± 2.7* 10.0 ± 0.6* 12.6 ± 0.7* 22.0 ± 0.7*
Lovastatin-treated EAN 5 72.0 ± 3.5* 11.6 ± 0.9* 15.4 ± 0.2* 31.6 ± 4.4*
Lovastatin + mevalonate EAN 5 71.6 ± 2.9* 12.4 ± 1.3* 14.0 ± 0.7* 34.8 ± 6.2*
Data shown are the means ± SEM of the indicated lipid expressed in mg/dL. *p < 0.01 when
compared with adjuvant controls, ANOVA with a Dunnett’s multiple comparison post hoc analysis.
HDL, high-density lipoprotein; LDL, low-density lipoprotein; EAN, experimental autoimmune
neuritis.
1270 J. P. Sarkey et al.
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
Lovastatin does not alter serum cholesterol content in
EAN
Compared with sera from naı̈ve or adjuvant control rats, sera
collected at peak of disease from vehicle-treated EAN rats
contained markedly reduced levels of total cholesterol, HDL
cholesterol, and triglyceride (Table 1). Calculated levels of
low-density lipoprotein cholesterol, however, were slightly
increased at peak of disease. Statins, including lovastatin,
belong to a group of potent HMG-CoA reductase inhibitors
that are clinically approved for cholesterol reduction. The
short-term duration of lovastatin treatment used in this study,
however, had no additional effects on the content of these
serum lipids (Table 1). These findings are consistent with
previous reports that short-term statin use does not alter
serum cholesterol levels in rodents (Krause and Newton
1995; Aktas et al. 2003; Gegg et al. 2005).
Effect of lovastatin on serum levels of ALT, AST, and CK
It is well established that long-term statin use in humans is
limited by associated myotoxic and hepatotoxic complica-
tions (Thompson et al. 2003; Cohen et al. 2006). To address
these concerns, we measured levels of the liver enzymes ALT
and AST as well as muscle enzyme CK in sera of naı̈ve,
adjuvant control-, vehicle-, and lovastatin-treated rats. The
levels of ALT, AST, or CK present in sera of lovastatin-treated
EAN rats collected at peak of disease were not significantly
different from that found in sera of vehicle-treated EAN rats
or that of naı̈ve or adjuvant controls (Table 2). These data
suggest that, at the doses used in this study (10–25 mg/kg/
day), a 7- to 14-day course of lovastatin-treatment does not
appear to be hepatotoxic or myotoxic to Lewis rats.
Mevalonate reverses the protective effects of lovastatin
Recent studies have shown that statins can alter immune cell
function independent of inhibition of HMG-CoA reductase
by directly interfering with leukocyte function-associated
antigen (LFA)-1 intercellular adhesion molecule-1 interaction
(Weitz-Schmidt et al. 2001). To determine if the protective
effects of lovastatin on EAN-induced peripheral nerve injury
are dependent on inhibition of HMG-CoA reductase, we
treated immunized rats with a combination of lovastatin and
mevalonate, the immediate metabolic product of this enzy-
matic reaction. Immunized rats receiving both mevalonate
and lovastatin developed clinical disease that was statistically
indistinguishable from vehicle-treated EAN rats (Fig. 1a).
Mevalonate also prevented lovastatin from protecting against
the development of peripheral nerve conduction deficits.
Evoked CMAP amplitudes were 20.3 ± 8.3% and
56.5 ± 20.4% of adjuvant controls or lovastatin-treated
immunized rats, respectively (see Fig. 2). Peripheral NCV
was slowed (33.2 ± 9.1 m/s) in mevalonate + lovastatin co-
treated immunized rats compared with adjuvant controls
(56.2 ± 5.8 m/s) or lovastatin-treated immunized rats
(55.6 ± 4.3 m/s). Mevalonate similarly diminished the pro-
tective effects of lovastatin on peripheral nerve morphology
(Figs 3c and f). Sciatic nerves harvested at peak of disease
from these rats yielded a histopathological score
(1.90 ± 0.10, n = 5) that was similar tovehicle-treated
immunized rats (2.30 ± 0.31, n = 5). Together, these data
strongly suggest that lovastatin treatment attenuates the
development of EAN-mediated peripheral nerve injury
through inhibition of HMG-CoA reductase.
Lovastatin suppresses T-cell responses in EAN
Through inhibition of HMG-CoA reductase, statins have been
shown to limit antigen-dependent T-cell proliferation and Th1
(pro-inflammatory) immune responses (Adamson and Green-
wood 2003). As shown in Fig. 4, splenocytes harvested from
vehicle-treated immunized rats at near peak of disease
exhibited a robust proliferative response that was enhanced
dose dependently by in vitro antigen (BPNM) stimulation
(6.5-fold maximal stimulation index, p < 0.01). Antigen
stimulation similarly elicited a measurable increase in pro-
inflammatory cytokine (IFN-c and TNF-a) expression
(Figs 4b and c). By comparison, splenocytes harvested from
lovastatin-treated immunized rats were significantly less
proliferative (p < 0.05, n = 4) and responded only poorly to
subsequent in vitro antigen (BPNM) stimulation (Fig. 4a).
Interestingly, antigen-stimulated pro-inflammatory cytokine
expression was not altered by in vivo lovastatin treatment
(Figs 4b and c). Although the expression of both IFN-c and
TNF-a appeared to be elevated following antigen stimulation,
these data were not significantly different compared with
antigen-stimulated splenocytes harvested from vehicle-trea-
Table 2 A short-term course of lovastatin-
treatment does not alter serum levels of
alanine transaminase, aspartate transami-
nase, or creatine kinase
Treatment group n ALT AST CK
Naı̈ve control 5 58.2 ± 3.8 107.8 ± 13.3 1361 ± 415
Adjuvant control 5 53.0 ± 1.7 102.8 ± 4.9 3545 ± 443
Vehicle-treated EAN 5 46.4 ± 1.5 122.6 ± 11.7 2348 ± 318
Lovastatin (10 mg/kg)-treated EAN 5 39.2 ± 1.3 143.6 ± 12.1 1664 ± 86
Lovastatin (25 mg/kg)-treated EAN 5 53.2 ± 8.4 161.8 ± 41.2 1489 ± 1354
Data shown are the means ± SEM of n observations of the indicated enzyme activity expressed in
U/L.
ALT, alanine transaminase; AST, aspartate transaminase; CK, creatine kinase; EAN, experimental
autoimmune neuritis.
Lovastatin attenuates experimental autoimmune neuritis 1271
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
ted rats. IL-12 (9.8 ± 1.5 pg/mL), IL-5 (11.4 ± 6.7 pg/mL),
and IL-10 (1006 ± 117 pg/mL) released from splenocytes
harvested from vehicle-treated immunized rats were unre-
sponsive to in vitro antigen stimulation and unaltered by
lovastatin. The resting and antigen-stimulated content of IL-4,
while assayed in this study, was found to be negligible.
Lovastatin reduces expression of MHC class II and B7
co-stimulatory antigen
To address the possibility that lovastatin may affect antigen-
mediated immune responses in EAN by altering the
expression of MHC class II (Kwak et al. 2000) or co-
stimulatory antigens (Youssef et al. 2002), we quantitated
the amount of MHC class II, B7-1, and B7-2 antigens
present on splenocytes in lovastatin-treated animals. We
found that, at peak of disease, splenocytes harvested from
lovastatin-treated immunized rats exhibited a modest (�20–
30%), but significant, reduction in the surface expression of
both MHC class II and B7-1 co-stimulatory antigens
present on CD11b/c-positive splenocyte APCs (Table 3).
A reduction in B7-2 was also noted, but this finding was
not statistically significant.
Lovastatin suppresses leukocyte migration
It is well established that the pathogenesis of EAN involves,
in part, cell-mediated autoimmunity (Tansey and Brosnan
1982; Linington et al. 1984; Taylor and Pollard 2001).
Perivascular infiltration of mononuclear cells are a charac-
teristic histologic hallmark of EAN (Lampert 1969; Strigard
et al. 1987). To determine if lovastatin alters leukocyte
migration in vivo, sciatic nerves from vehicle- or lovastatin-
treated immunized rats were immunostained for the presence
of CD68+ macrophage, CD4+, or CD8+ lymphocyte infil-
trates. We found that lovastatin reduced both macrophage
and lymphocyte migration into peripheral nerve of EAN rats
(Figs 5a and b). Previous studies have shown that statins may
prevent activated leukocytes from entering immune-privi-
leged sites by directly inhibiting transendothelial migration
(Greenwood et al. 2003). Treating peripheral nerve endo-
thelial cell monolayers in vitro with lovastatin produced a
marked reduction in the migration of antigen (BPNM)-
activated splenocytes (Fig. 5c). Together, these findings
suggest that lovastatin protects against the development of
EAN-induced peripheral nerve injury by a mechanism that
limits the transendothelial migration of autoreactive leuko-
cytes into peripheral nerve.
Discussion
The major finding of this study is that a short-term course of
parenterally administered high-dose lovastatin markedly
0 
2000 
4000 
6000 
8000 
0 1 10 100 
EAN + vehicle 
EAN + Lov (10 mg/kg) 
* ** 
BPNM (µg/mL)
(a) 
(b) 
[3
H
]T
hy
m
id
in
e 
(d
pm
) 
Vehicle Lovastatin
0
5
10
15
20
Vehicle Lovastatin
T
N
F
α 
(p
g/
m
L
)
0
1000
2000
3000
4000
5000
IF
N
γ 
(p
g/
m
L
)
*
*
*
*
Fig. 4 Lovastatin attenuates antigen-stimulated splenocyte prolifer-
ation without altering pro-inflammatory cytokine production. Immunized
rats were treated daily with 50% dimethylsulfoxide (vehicle) or lovast-
atin (10 mg/kg) prior to disease onset as indicated in Fig. 1a and
spleens harvested on day 21 post-immunization. (a) Antigen-stimulated
proliferation of cultured splenocytes was quantitated as described un-
der Materials and methods. Data shown are the means ± SEM from
four rats per treatment group. **p < 0.001; *p < 0.05, relative to vehicle-
treated controls (open bars); unpaired ANOVA with Bonferroni multiple
comparison post-test. (b, c) Resting (open bars) and antigen (BPNM)-
stimulated (shaded bars) levels of interferon-c (IFN-c) (b) and tumor
necrosis factor-a (TNF-a) (c) present in the media of cultured spleno-
cytes harvested from vehicle- or lovastatin-treated immunized rats.
Data shown are the means ± SEM from five rats per treatment group.
*p < 0.05, relative to un-stimulated resting controls, Student’s t-test.
Table 3 Effect of in vivo lovastatin treatment on CD11b/c-positive
splenocyte cell-surface expression of MHC II, B7-1, and B7-2 antigens
Treatment group n MHC II CD80 (B7-1) CD86 (B7-2)
Vehicle-treated EAN 5 55.2 ± 2.1 6.7 ± 0.6 28.0 ± 3.4
Lovastatin (10 mg/kg)-
treated EAN
5 43.5 ± 2.2* 4.5 ± 0.4* 22.2 ± 3.0
Splenocytes were prepared from vehicle- or lovastatin-treated
immunized rats at peak of disease and the percent expression of the
indicated surface antigens on CD11b/c-positive cells was determined
by flow cytometric analysis as described under Materials and meth-
ods. Data shown are the means ± SEM of n observations. *p < 0.01,
compared with vehicle-treated EAN rats, Student’s t-test.
EAN, experimental autoimmune neuritis; MHC, major histocompati-
bility complex.
1272 J. P. Sarkey et al.
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
inhibits the development and progression of EAN without
eliciting hepatotoxic or myotoxic complications. Independent
of cholesterol reduction, lovastatin treatment was found to
protect against EAN-induced peripheral nerve conduction
deficits and morphologic nerve injury by a mechanism that
limits the proliferation and transendothelial migration of
Vehicle 
(a) 
(b) 
(c) 
CD68+ CD4+ 
Lova
0
50
100
150
200
Vehicle Lovastatin 
* 
0
50
100
150
200
Vehicle Lovastatin 
* 
0 
50 
100 
150 
200 
Vehicle Lovastatin 
* 
L
ym
ph
oc
yt
es
 
(C
D
8+
 c
el
ls
 p
er
 0
.5
 m
m
2 
 fi
el
d)
L
ym
ph
oc
yt
es
 
(C
D
4+
 c
el
ls
 p
er
 0
.5
 m
m
2 
 fi
el
d)
M
ac
ro
ph
ag
es
 
(C
D
68
+ 
ce
lls
 p
er
 0
.5
 m
m
2 
 fi
el
d)
0
20
40
60
80
100
120* * 
* 
0 0.1 1.0 10.0
Lovastatin (µmol/L)
Sp
le
no
cy
te
 m
ig
ra
ti
on
(%
 o
f 
co
nt
ro
l)
Fig. 5 Lovastatin suppresses leukocyte
transendothelial migration. Immunized rats
were treated daily with 50% dimethylsulf-
oxide (vehicle) or lovastatin (Lova; 10 mg/
kg) prior to disease onset as indicated in
Fig. 1a and sciatic nerves harvested on day
21 post-immunization. (a) Shown are rep-
resentative longitudinal sections of nerves
immunostained for the presence of CD68+
macrophages (left panels, n = 5) or for
CD4+ lymphocytes (right panels, n = 5).
Scale bar: 50 lm. Arrows, immunostained
cells. (b) Quantitative comparison of leuko-
cyte infiltration seen in (a) between vehicle-
and lovastatin-treated immunized rats. Data
shown are the means ± SEM from five rats
per treatment group.*p < 0.01, Student’s
t-test. (c) Effect of lovastatin on in vitro
migration of splenocytes through a periph-
eral nerve endothelial monolayer. Data
shown are the means ± SEM, n = 5.
*p < 0.0001; one-way ANOVA with Dunnett’s
multiple comparison post-test.
Lovastatin attenuates experimental autoimmune neuritis 1273
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
autoreactive leukocytes. We postulate here that a short-term
application of high-dose statins may prove clinically useful
in the management of inflammatory demyelinating peripheral
nerve diseases, including some clinical subgroups of GBS.
Currently, statins are prescribed to more than 25 million
people worldwide and have emerged as the leading thera-
peutic option for treating hypercholesterolemia and reducing
associated cardiovascular morbidity and mortality. All
statins, whether naturally occurring or synthetic, reduce
endogenous cholesterol levels in patients by competitive
inhibition of HMG-CoA reductase, the rate-limiting enzyme
in cholesterol biosynthesis. The therapeutic effect of statins,
until recently, was believed to be directly due to its potent
cholesterol-lowering properties. It is now recognized, how-
ever, that not all the benefits of statins are attributable to
cholesterol reduction alone. A number of pleiotropic effects
have now been described for both naturally occurring and
synthetic statins, including important anti-inflammatory
(Katznelson and Kobashigawa 1995; Kobashigawa et al.
1995) and neuroprotective properties (Stanislaus et al. 1999;
Stuve et al. 2003; Paintlia et al. 2005). As a result, statins are
now being considered as potential therapeutic agents for the
management of a wide variety of inflammatory diseases,
including several neurologic disorders as disparate as
ischemic stroke, Alzheimer’s disease, and multiple sclerosis
(Menge et al. 2005; Greenwood et al. 2006).
The use of statins as an adjunctive therapy is not, however,
without controversy. Infrequent, but potentially severe and
life-threatening, side effects of prolonged statin therapy have
been reported and include peripheral sensory neuropathy
(Gaist et al. 2002), rhabdomyolysis (Thompson et al. 2003),
and hepatotoxic complications (Cohen et al. 2006). In this
study, where a short-term duration (7–14 days) of high-dose
lovastatin was evaluated, we found no evidence at peak of
disease of hepatotoxic (as determined by serum ALT and
AST levels) or myotoxic (serum CK) complications. These
findings raise the possibility that high-dose statins adminis-
tered over a short-term course may minimize the risk of
chronic toxic side effects commonly associated with these
potent HMG-CoA reductase inhibitors.
The dose of statins approved for the long-term manage-
ment of hypercholesterolemia (�0.5–1 mg/kg/day) in hu-
mans are only modestly effective at altering immune
responses in experimental animal models of autoimmune
disease. In one study, however, statins were shown to be
effective even at doses that are comparable with therapeutic
use in humans (Youssef et al. 2002). On the other hand, the
dose and route of statin administration required to signifi-
cantly attenuate immune responses often differ with the type
of autoimmune disease induced, the statin used, and the
species and strain of animal utilized (Greenwood et al.
2006). In a recent study by Gegg et al. (2005), mice treated
parenterally (20 mg/kg/day) with lovastatin exhibited plasma
concentrations of lovastatin hydroxy acid that was compar-
able with therapeutic concentrations reported in human
studies, suggesting that the pharmacokinetic properties of
statins may differ substantially between humans and rodents
(Black et al. 1998; Stern et al. 2000). Despite these differ-
ences, encouraging results from in vivo animal models and
clinical observational studies (Vollmer et al. 2004; Silva
et al. 2006) continue to emerge supporting a beneficial role
of statins for the treatment of certain neurologic disorders
including, as shown in the present study, inflammatory
demyelinating peripheral nerve disease.
Several biological properties of statins might influence the
development or progression of EAN. Statin-dependent inhi-
bition of HMG-CoA reductase leads to changes in the post-
translational isoprenylation of proteins that are associated
with several important immune cell functions (Liao 2002;
Wozniak et al. 2005). One such function includes the homing
and migration of activated leukocytes into the neural paren-
chyma. By limiting isoprenoid synthesis, lovastatin has been
shown to reduce the migration of autoreactive lymphocytes
across the blood–brain barrier in experimental autoimmune
encephalomyelitis (EAE), an animal model of multiple
sclerosis (Stanislaus et al. 2002). Statins have also been
shown in vitro to inhibit the migration of lymphocytes through
blood–brain barrier-derived endothelial cell monolayers
(Greenwood et al. 2003). Consistent with a role of statins
affecting leukocyte trafficking in the CNS, we observed that
lovastatin significantly reduced macrophage and lymphocyte
infiltration into sciatic nerves of immunized rats. In addition,
treating peripheral nerve endothelial cell monolayers in vitro
with lovastatin significantly inhibited the migration of auto-
reactive splenocytes. This demonstrates, for the first time, the
ability of statins to inhibit leukocyte migration across
endothelial cells of the blood–nerve barrier in EAN.
Statin-dependent inhibition of leukocyte migration across
the peripheral nerve endothelial barrier was most likely due
to inhibition of HMG-CoA reductase independent of cho-
lesterol reduction. Co-administration of mevalonate, the
immediate metabolic product of HMG-CoA reductase,
prevented the protective effects of lovastatin. Short-term
administration of lovastatin, however, failed to produce
measurable changes in serum cholesterol content, consistent
with previously reported studies (Aktas et al. 2003; Gegg
et al. 2005). Immunized rats co-treated with lovastatin and
squalene (2 mg/kg), a metabolic precursor of cholesterol, had
no effect on the protective effects of lovastatin (data not
shown). Together, these data support a cholesterol-independ-
ent mechanism of statin-dependent protection against EAN-
induced peripheral nerve injury.
In addition to limiting leukocyte trafficking within the
PNS, statins may also inhibit leukocyte proliferation by
altering the inducible expression of MHC class II antigens
or co-stimulatory molecules on APCs (Kwak et al. 2000;
Youssef et al. 2002). Consistent with the effects of statins
on EAE (Greenwood et al. 2006), splenocytes harvested
1274 J. P. Sarkey et al.
� 2006 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1265–1277
No claim to original US government works
from lovastatin-treated immunized rats in this study
exhibited a reduced ability to proliferate in response to
in vitro antigen stimulation. We also observed a modest
(�20–30%), but significant, reduction in the surface
expression of both MHC class II and B7-1 co-stimulatory
antigens present on APCs. These findings support the thesis
that statinsmay affect autoreactive leukocyte proliferation
by a mechanism that alters surface expression of MHC
class II or co-stimulatory molecules on APCs. Alternat-
ively, statins may affect leukocyte proliferation by disrupt-
ing isoprenoid synthesis, thereby limiting the functional
maturation of small G proteins, including Ras (Ghittoni
et al. 2005). Consistent with this possibility, we observed a
modest, but significant, decrease (31.0 ± 6.9%; p < 0.03,
n = 5) in the content of membrane-associated Ras in
splenocytes harvested from lovastatin-treated immunized
rats. Reduced clonal expansion of autoreactive leukocytes
by statins, together with limiting transendothelial leukocyte
trafficking, would be expected to ameliorate clinical signs
of EAN.
Splenocytes harvested from animals with EAE show a
statin-dependent bias toward Th2 lymphocyte profiling
(Stanislaus et al. 2002; Youssef et al. 2002; Aktas et al.
2003; Gegg et al. 2005; Dunn et al. 2006). As it is well
established that EAN is also a Th1-mediated disease, these
findings raise the possibility that lovastatin may protect
immunized rats against peripheral nerve injury by similarly
inducing a bias toward Th2 cytokines. In contrast, however,
we observed that in vivo lovastatin treatment did not
attenuate resting or antigen-stimulated production of pro-
inflammatory (IFN-c, TNF-a) or anti-inflammatory (IL-5, IL-
10) cytokines from harvested splenocytes. By comparison,
parenteral treatment of experimental autoimmune uveitis
with lovastatin elevated pro-inflammatory TNF-a while
reducing both IFN-c and IL-10 (Gegg et al. 2005). Treat-
ment of experimental systemic lupus erythematosus with
high-dose atorvastatin did not alter either Th1 or Th2
cytokines (Lawman et al. 2004). These findings strengthen
the idea that there are potential differential effects of statins
on different inflammatory autoimmune disorders. A short-
term course of lovastatin treatment, as used in this study,
does not substantially alter Th1/Th2 lymphocyte profiling in
EAN. We cannot, however, rule out the possibility of
localized changes in Th1/Th2 balance occurring within other
affected lymphoid organs.
In summary, we show that a short-term course of high-
dose lovastatin attenuates the development and progression
of EAN-induced peripheral nerve injury by a mechanism that
limits the proliferation and transendothelial migration of
autoreactive leukocytes. We postulate that a short-term
course of high-dose statin intervention may be of therapeutic
interest for the treatment of inflammatory demyelinating
peripheral nerve disorders, including GBS.
Acknowledgements
The authors wish to thank Dr Lisa Halliday for assistance procuring
bovine cauda equina, Dr Matthew Sorenson for assistance with Bio-
plex system, and Ms Linda McCann and Mr Thomas Hoagland for
assistance with the determination of cholesterol, triglyceride, ALT,
AST, and CK in serum samples. J. P. Sarkey was supported by a
fellowship from the Arthur J. Schmitt Foundation. This work was
supported by grants from the Department of Veteran Affairs,
Veterans Health Administration and the Potts Foundation (Loyola
University Chicago).
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