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ORIGINAL PAPER
Doxycycline Attenuates Peripheral Inflammation in Rat
Experimental Autoimmune Neuritis
Chenju Yi •Zhiyuan Zhang •Wei Wang •
Caroline Zug •Hermann J. Schluesener •
Zhiren Zhang
Accepted: 28 May 2011 / Published online: 8 June 2011
ÓSpringer Science+Business Media, LLC 2011
Abstract Experimental autoimmune neuritis (EAN) is a
T cell-mediated autoimmune inflammatory demyelinating
disease of the peripheral nervous system and widely-used
animal model of human inflammatory demyelinating
polyradiculoneuropathies. Doxycycline is a well-known
antibiotic and has been reported to have neuroprotective
and anti-inflammatory effects. Here we investigated the
effects of doxycycline on rat EAN. Therapeutic treatment
with doxycycline (40 mg/kg body weight daily from the
Day 9 to Day 14 post immunization) significantly attenu-
ated the severity of EAN, decreased inflammatory infil-
tration of macrophages, B- and T-cells and demyelination
in sciatic nerves of EAN rats. Pro-inflammatory molecules
including matrixmetalloproteinase-9, inducible nitric oxide
synthase and interleukin-17 were greatly decreased in sciatic
nerves by administration of doxycycline as well. Taken
together, our data showed that doxycycline could effec-
tively suppress the peripheral inflammation to improve
outcome of EAN, which suggests that doxycycline may be
considered as a potential candidate of pharmacological
treatment for neuropathies.
Keywords Doxycycline EAN Inflammation
Sciatic nerves
Introduction
Experimental autoimmune neuritis (EAN) is an autoantigen-
specific T-cell-mediated inflammatory peripheral nervous
system (PNS) demyelinating animal model [1]. EAN can be
induced in susceptible animals by active immunization with
whole PNS myelin or its components, the proteins P2 or P0,
as well as their immunogenic peptides [2]. EAN is patho-
logically characterized by breakdown of the blood-nerve
barrier, infiltration of reactive immune cells, local inflam-
mation and demyelination in the peripheral nervous. EAN
shares many clinical, electrophysiological and immunolog-
ical features of human acute inflammatory demyelinating
polyradiculoneuropathy (AIDP) and has been widely used
as an animal model to study disease mechanism and therapy
of AIDP. Treatments of AIDP include plasma exchange,
intravenous immunoglobulin or supportive management
such as intensive care and respiratory assistance. But all of
these treatments are unsatisfying [1], and new therapeutic
options are therefore needed.
Doxycycline is a tetracycline antibiotic. Besides its
broad-spectrum antibiotic activity, doxycycline has been
shown to display neuroprotective and anti-inflammatory
properties in animal models of global and focal cerebral
ischemia [3,4], multiple sclerosis [5], Parkinson’s disease
[6]; and in clinical trials of Alzheimer’s disease, lym-
phangioleiomyomatosis, osteoarthritis and others [7].
Neuroprotective and anti-inflammatory effects of doxycy-
cline in these disorders are associated with inhibition of
matrix metalloproteases [8,9], depression of oxygen radi-
cal release from polymorphonuclear neutrophils [10],
C. Yi and Z. Zhang contributed equally to this work.
C. Yi Z. Zhang C. Zug H. J. Schluesener Z. Zhang (&)
Institute of Brain Research, University of Tuebingen,
Calwer Street 3, 72076 Tuebingen, Germany
e-mail: zhangzhiren@yahoo.com
C. Yi W. Wang
Department of Neurology, Tongji Hospital, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan, People’s Republic of China
Z. Zhang
Institute of Immunology, Third Military Medical University
of PLA, Chongqing, People’s Republic of China
123
Neurochem Res (2011) 36:1984–1990
DOI 10.1007/s11064-011-0522-2
inhibition of inducible nitric oxide synthase and inter-
leukin-1b-converting enzyme [3], reduction of cleaved
caspase-3 protein expression and decrease of microglial
activation [11,12].
Doxycycline is commonly used as an antibiotic and its
toxicity has been characterized in detail [13]. Additionally,
the high lipophilicity of doxycycline allows it to diffuse
into the central and peripheral nervous system (CNS and
PNS) at therapeutically effective levels [14]. Therefore, the
proven reliability and safety of doxycycline suggest its
potential prospect as an effective and cheap treatment of
human inflammatory neuropathies. In our present investi-
gation, we determined the potential protective effect of
doxycycline on rat EAN.
Experimental Procedure
Animal Experiments
Male Lewis rats (8–10 wee ks old, 200–250 g, Charles River,
Sulzfeld, Germany) were housed with equal daily periods of
light and dark and free access to food and water. All proce-
dures were performed in accordance with the published
International Health Guidelines under a protocol approved
by the local Administration District Official Committee. All
efforts were made to minimize the number of animals and
their suffering. EAN was induced by subcutaneous injection
at the base of the tails with 100 ll of an inoculum containing
100 lg of synthetic neuritogenic P2
53–78
peptide (Gene
Script Corporation, Scotch Plains, NJ, USA). The peptide
was dissolved in phosphate-buffered saline (PBS) (2 mg/ml)
and then emulsified with an equal volume of complete Fre-
und’s adjuvant (CFA) containing 2 mg/ml Mycobacterium
tuberculosis to get a final concentration of 1 mg/ml. The
severity of EAN was scored daily as follows: 0-normal,
1-reduced tonus of tail, 2-limp tail, impaired righting,
3-absent righting, 4-gait ataxia, 5-mild paresis of the hind
limbs, 6-moderate paraparesis, 7-severe paraparesis or
paraplegia of the hind limbs, 8-tetraparesis, 9-moribund,
10-death. As soon as first neurological signs were observed,
all the immunized rats were randomly divided into two
groups and were treated with doxycycline and control,
respectively. For therapeutic treatment, doxycycline (Sigma,
St. Louis, MO, USA; 40 mg/kg body weight in 1 ml PBS)
was intraperitoneally injected once daily from the Day 9 to
Day 14 post immunization. The PBS control group received
the same volume of PBS.
LFB Staining and Immunohistochemistry
Luxol fast blue (LFB) staining was applied to show
myelin and to evaluate demyelination of sciatic nerves.
For LFB staining, histological changes between doxy-
cycline and PBS treated EAN rats were compared by an
established semi-quantitative method. Briefly, four cross-
sections from sciatic nerves of both sides of EAN rats
were analyzed. All perivascular areas present in cross-
section were evaluated by two observers unaware of
treatment, and the degree of pathological alteration was
graded semi-quantitatively by the following scale:
0=normal perivascular area; 1 =mild cellular infiltra-
tion adjacent to the vessel; 2 =cellular infiltration plus
demyelination in immediate proximity to the vessel;
3=cellular infiltration and demyelination throughout the
section. Results were given as means of histological
scores [15]. Results were calculated as arithmetic means
of positive cells per square millimeter and standard
errors of means (SEM).
Immunohistochemistry (IHC) was performed on 3 lm
paraffin-embedded sections using antibodies serially to
evaluate local inflammation, cellular infiltration and
demyelination in sciatic nerves: ED-1 (1:100; Serotec,
Oxford, UK) to detect activated microglia/macrophages,
OX22 (1:200; Serotec, Oxford, UK) predominantly for B
cells or CD3 (1:50; Serotec, Oxford, UK) predominantly
for T-lymphocytes, Interleukin-17 (IL-17; 1:100; Santa
Cruz Biotechnology, Santa Cruz, CA, USA), iNOS (1:100;
Serotec, Oxford, UK) and MMP9 (matrix metalloprotein-
ase-9; 1:500; Neuromics, Edina, MN, USA). After
dewaxing, sections were boiled (in an 850 W microwave
oven) for 15 min in citrate buffer (2.1 g citric acid
monohydrate/L, pH6) (Carl Roth, Karlsruhe, Germany).
Endogenous peroxidase was inhibited by 1% H
2
O
2
in pure
methanol (Merck, Darmstadt, Germany) for 15 min. Sec-
tions were incubated with 10% normal pig serum (Bio-
chrom, Berlin, Germany) to block non-specific binding of
immunoglobulins and then with the primary antibodies
overnight at 4°C. Antibodies binding to tissue sections
were visualized with secondary biotinylated antibodies
(rabbit anti-mouse or rabbit anti-goat) (1:400; DAKO,
Hamburg, Germany). Subsequently, sections were incu-
bated with a Streptavidin–Avidin–Biotin complex (DAKO,
Hamburg, Germany), followed by development with
diaminobenzidine (DAB) substrate (Fluka, Neu-Ulm,
Germany). Finally, sections were counterstained with
hemalum. As negative controls, the primary antibodies
were omitted.
After immunostaining, sections from doxycycline and
PBS control groups were examined by light microscopy
and the numbers of ED-1
?
,OX22
?
, CD3
?
, IL-17
?
,
iNOS
?
and MMP-9
?
cells were counted. Positively
stained cell counting based on IHC results has been well
developed to semi-quantify protein expression [16]. The
numbers of positively stained cells were counted by two
investigators independently and only positive cells with
Neurochem Res (2011) 36:1984–1990 1985
123
the nucleus at the focal plane were counted. The sections
were randomly numbered and observers were not aware
of the time points and treatments. To evaluate positive
cell numbers in sciatic nerves, four cross-sections for
each rat were evaluated. Microphotos of the whole sci-
atic nerve cross-sections were taken under 200 9mag-
nification using Nikon Coolscope (Nikon, Du
¨sseldorf,
Germany) and only positive cells with the nucleus at the
focal plane were counted. Areas of sciatic nerve cross-
sections were measured on the same pictures using
software MetaMorph Offline 7.1 (Molecular Devices,
Toronto, Canada).
Statistical Analysis
Statistical analysis was performed by one-way ANOVA
followed by Dunnett’s multiple comparison test (Graph
Pad Prism 4.0 software). For all statistical analyses, sig-
nificance levels were set at P\0.05.
Results
Effects of Doxycycline on Pathological Scores
and Body Weight of EAN Rats
EAN was induced by subcutaneous injection of P2 peptide.
The first neurologic sign (reduced tonus of tail) of EAN
rats was observed at day 9 (mean clinical score ±SEM:
0.20 ±0.20). For therapeutic treatment, doxycycline or
PBS (PBS control group) was injected once daily from the
onset of neurological signs (day 9–14 post immunization).
The neurologic severity of EAN increased fast in the
control group with a maximal score at day 15 (mean
clinical score ±SEM: 6.8 ±0.20). However, doxycycline
therapeutic treatment suppressed severity of EAN (maxi-
mal mean score at day 15: 4.0 ±0.32, P\0.05 compared
to PBS control) from day 12 to 15 (P\0.05, compared to
respective control) (Fig. 1a).
Progressive weight loss during onset of EAN is another
characteristic feature of this disease, which correlates with
Fig. 1 Doxycycline reduced pathological scores and mean histolog-
ical scores. EAN was induced by subcutaneous injection of synthetic
neuritogenic P2 peptide (100 ug) and CFA into the base of the tails.
Doxycycline or PBS (five rats each, 40 mg/kg body weight in 1 ml
PBS) were injected once daily starting from 9th day until 14th day
after immunization. Clinical scores were taken from day 3 after
immunization. aDoxycycline treatment significantly decreased
neurologic severity of EAN. bDoxycycline suppressed histopathol-
ogical alterations in sciatic nerves of EAN rats. 15 days after
immunization, sciatic nerves of doxycyline treated and PBS control
rats were taken and used for LFB staining, followed by hematoxylin
counterstaining. Representative micrographs for PBS or doxycycline
treated EAN rats are shown in cand d, respectively. Means of
histological scores were calculated as described in ‘‘Experimental
Procedures’’ . Bar figure shows that doxycycline treatment signifi-
cantly reduced mean histological scores as compared to PBS controls.
The unpaired ttest was performed (Graph Pad Prism 4.0 for
Windows). * P\0.05 compared to the PBS control
1986 Neurochem Res (2011) 36:1984–1990
123
the severity of EAN. A progressive weight loss was observed
during the onset of EAN from Day 9 to Day 15 in PBS treated
EAN rats. In contrast, the weight of doxycycline-treated rats
increased slightly progressively after day 12. Significant
differences of body weight between PBS- and doxycycline-
treated EAN rats from day 12 to day 15 was proven by one-
way ANOVA followed by Dunnett’s multiple comparison
test (P\0.05)(data not showed). Taken together, these
results indicated a significantly reduced disease severity in
doxycycline-treated EAN.
Effects of Doxycycline on Demyelination and Cell
Infiltration in EAN Sciatic Nerves
Sciatic nerves were taken from doxycycline-treated and
PBS control EAN rats (n=5) at Day 15 and were
analyzed histologically. LFB staining demonstrated a
significant decreased degree of perivascular demyelina-
tion and inflammatory cell infiltration in EAN rats
treated with doxycycline (mean histological score 1.45 ±
0.09) (Fig. 1b, d), as compared to the PBS control group
(mean histological score 2.13 ±0.11) (Fig. 1b, c),
P\0.05.
Distinct infiltration of different types of inflammatory
cells into sciatic nerves was further analyzed by IHC.
Dense inflammatory accumulation of macrophages
(ED-1
?
) (Fig. 2a), T cells (CD3
?
) (Fig. 2b), B cells
(OX22
?
) (Fig. 2c) were seen in sciatic nerves of rats from
the PBS control group at Day 15 and the most dominant
cell population were macrophages, with a mean density of
652.2 ±77.26 cells/mm
2
(Fig. 2g). Doxycycline treatment
significantly decreased inflammatory cell accumulation
Fig. 2 Doxycycline suppressed macrophage, T cell and B cell
infiltration into sciatic nerves of EAN rats. 15 days after immuniza-
tion, sciatic nerveswere analyzed by immunohistochemstry. ED-1
(aand d) immunostaining was used for macrophages, CD3 (band
e) for T cells and OX22 (cand f) for B cells. Representative
micrographs from doxycycline treated and PBS control EAN rats are
shown in (a–c) and (b–d), respectively. Infiltration of macrophages,
T- and B-cells into sciatic nerves was further semi-quantified as
indicated in ‘‘Experimental Procedures’’ and the figures show the
quantified results (g–i). Doxycycline treatment significantly reduced
infiltration of macrophages, T cell and B cells into EAN sciatic nerves
(doxy: doxycycline treatment). The unpaired ttest was performed to
compare the differences (Graph Pad Prism 4.0 for Windows).
*P\0.05 compared to their respective control
Neurochem Res (2011) 36:1984–1990 1987
123
(P\0.05, compared to PBS control, respectively)
(Fig. 2d–i).
Effects of Doxycycline on Levels of IL-17, iNOS
and MMP-9 in EAN Sciatic Nerves
We further studied cellular expression of several important
inflammatory cytokines/molecule. In sciatic nerves from
control EAN rats receiving injections of PBS only, IL-17,
iNOS and MMP9 were observed in aggregated immune
cells. Doxycycline treatment strikingly reduced locally
accumulated IL-17
?
(Fig. 3a, d, g), iNOS
?
(Fig. 3b, e, h)
and MMP9
?
(Fig. 3c, f, i) cells in EAN sciatic nerves
(P\0.05, compared to PBS controls separately)
Discussion
Here we have studied the therapeutic effects of doxycy-
cline on EAN, which is the prime animal model for AIDP
and useful for investigating therapeutic approaches. Ther-
apeutic treatment with doxycycline significantly reduced
neurologic severity of EAN through reducing local demy-
elination, suppression of local inflammatory cell infiltration
and decreasing expression of pro-inflammatory molecules
MMP-9, iNOS and IL-17 in sciatic nerves.
As a member of the tetracycline class of antibiotics,
doxycycline has been shown interesting pleiotropic prop-
erties, such as neuroprotective and anti-inflammatory
effects. In vitro and in vivo data have suggested that
doxycycline inhibited inflammation by modulating cellular
activation and subsequent release of cytokines, chemo-
kines, lipid mediators of inflammation, MMPs and nitric
oxide [14]. In our EAN models, doxycycline greatly sup-
pressed infiltration of T cells, B cells and macrophages into
peripheral nerves and thus greatly reduced the patholog-
icalinfiltration of reactive leucocyte into the PNS [17].
Doxycycline inhibited MMP expression in sciatic nerves
to reduce immune cell infiltration in sciatic nerves of EAN
rats. MMPs, particularly MMP-9, could participate in the
disruption of the BNB, breakdown of the myelin sheath,
the release of TNF-a, and finally facilitate leukocyte
invasion into the PNS [18]. Previous studies have shown an
increased expression of MMP-9 in inflamed peripheral
nerves in EAN [19]. In the nervous system, aberrant
expression of MMPs may support disease activity by
converting pro-forms of several inflammatory molecules,
such as TNF-a, into their active forms, resulting in the
Fig. 3 Doxycycline suppressed
accumulation of IL-17, iNOS
and MMP-9 in sciatic nerves of
EAN rats. 15 days after
immunization, sciatic nerves of
both groups were taken and
used for immunohistochemical
staining of IL-17, iNOS and
MMP-9 (PBS control: a,band
cseparately; Doxycyline group:
d,eand fseparately).
Representative
immunohistochemical
micrographs showed that
doxycycline significantly
reduced the expression of IL-17,
iNOS and MMP-9 in sciatic
nerves. g–ibar figures show
quantified results. The unpaired
ttest was performed to compare
the differences between
doxycycline treated and PBS
control EAN rats (Graph Pad
Prism 4.0 for Windows).
** P\0.05, compared to their
respective control
1988 Neurochem Res (2011) 36:1984–1990
123
propagation of inflammation [20]. In addition, MMPs were
reported to induce the degradation of myelin or axonal
injury after injection into the brain [21], and fragments of
MMP-mediated digestion of myelin basic protein are
encephalitogenic when injected into mice. Thereby, a cas-
cade of demyelinating and pro-inflammatory events is
generated in the nervous system as a result of aberrant MMP
expression [22]. Doxycycline not only inhibits the enzy-
matic activity of MMPs, but also reduces the expression of
several other MMP family members [23]. Here, our data
showed that doxycycline reduced MMP-9 level in sciatic
nerves of EAN rats, which could not only inhibit leucocyte
infiltration but also diminish their effects on demyelination.
Doxycycline could also attenuate inflammatory cyto-
kines in sciatic nerves. Cytokines are produced and
released by many cell types and regulate inflammation and
immunity. Pro-inflammatory cytokines, such as IL-17
produced by Th cells, augment both inflammation and
subsequent immune responses [24]. IL-17 stimulates pro-
duction of IL-6, nitric oxide and prostaglandin E2 to
amplify local inflammation, mediates chemotaxis of neu-
trophils and monocytes to sites of inflammation and aug-
ments the induction of co-stimulatory molecules such as
ICAM-1 to support T cell activation [25,26]. Similar to
inflammatory cytokines, iNOS is up-regulated in EAN and
known to play important roles in inflammatory progression
of disease [27,28]. iNOS functions to produce nitric oxide
which possesses pro-inflammatory property including
vasodilation, oedema, cytotoxicity and mediates cytokine-
dependent processes that can result in tissue destruction
[29]. In EAN, upregulation of iNOS was reported and was
particularly related to pathogenesis of PNS cell-mediated
demyelination and even axonal damage [30]. In our study,
doxycycline reduced lesional iNOSs, which may inhibit
activation of immune cells, and be an important additional
part of doxycycline’s anti-inflammatory effect. Interest-
ingly, inhibition of iNOS expression is considered to be a
major mechanism of doxycycline [14]. Accumulated data
propose several potential mechanisms of inhibitory activity
of doxycycline on macrophage activity, but the exact
mechanisms are not clear yet. One of the potential mech-
anisms is inhibition of the expression of iNOS, resulting
in reduced release of NO and NO induced phosphorylation
of p38 MAPK (mitogen-activated protein kinase) [23].
Taken together, doxycycline greatly attenuated peripheral
inflammation and decreased local expression/release of
multiple inflammatory cytokines/molecules.
It is well known that another tetracycline, minocycline,
also has anti-inflammatory and neuroprotective effects
similar to doxycycline, as we had reported previously [31].
However, minocycline is also known for its side effects,
such as vestibular toxicity [32] and effects on bone and
teeth formation. Recently, decreased tolerability of
minocycline has also been raised as a concern during a
clinical trial of Parkinson’s Disease [33]. In contrast,
doxycycline has the least toxic side effects among tetra-
cyclines [34]. Although doxycycline and minocycline have
similar chemical structures, they have been reported to
exhibit differences in patterns of neuroprotection in some
models of cerebral ischemia [3,11,35] and in antioxidant
activity [36,37]. Additionally, doxycycline is a more
potent inhibitor of MMPs [37]. These data suggest that
doxycyline has more advantages in clinical applications.
In conclusion, doxycycline is inexpensive and easily
obtainable, could effectively suppress peripheral inflam-
mation to improve outcome in EAN rats. Our results sug-
gest that doxycycline should be considered a potential
candidate toward the therapeutic strategy for autoimmune
neuropathies.
Acknowledgments Chenju Yi gratefully acknowledged China
Scholarship Council. This investigation was partly supported by
National Nature Science Foundation of China (No: 81070954).
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