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The Journal of Neuroscience, March 15, 2002, 22(6):2246-2254
Microglial Activation and  -Amyloid Deposit Reduction
Caused by a Nitric Oxide-Releasing Nonsteroidal
Anti-Inflammatory Drug in Amyloid Precursor Protein Plus Presenilin-1
Transgenic Mice
Paul T.
Jantzen1,
Karen E.
Connor1,
Giovanni
DiCarlo1,
Gary L.
Wenk3,
John L.
Wallace4,
Amyn M.
Rojiani2,
Domenico
Coppola2,
Dave
Morgan1, and
Marcia N.
Gordon1
Departments of 1 Pharmacology and
2 Interdisciplinary Oncology, Alzheimer's Research
Laboratory, University of South Florida, Tampa, Florida 33612, 3 Division of Neural Systems, Memory, and Aging, University
of Arizona, Tucson, Arizona 85724, and 4 Department of
Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta,
T2N 4N1, Canada
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ABSTRACT |
3-4-(2-Fluoro- -methyl-[1,1'-biphenyl]-4-acetyloxy)-3-methoxyphenyl]-2-propenoic
acid 4-nitrooxy butyl ester (NCX-2216), a nitric oxide (NO)-releasing
derivative of the cyclooxygenase-1-preferring nonsteroidal
anti-inflammatory drug (NSAID) flurbiprofen, dramatically reduced both
 -amyloid (A) loads and Congo red staining in doubly transgenic
(Tg) amyloid precursor protein plus presenilin-1 mice when
administered at 375 ppm in diet between 7 and 12 months of age. This
reduction was associated with a dramatic increase in the number
of microglia expressing major histocompatibility complex-II antigen, a marker for microglial activation. In contrast, ibuprofen at
375 ppm in diet caused modest reductions in A load but not Congo red
staining, suggesting that the effects of this nonselective NSAID were
restricted primarily to nonfibrillar deposits. We detected no effects
of the cyclooxygenase-2-selective NSAID celecoxib at 175 ppm on amyloid
deposition. In short-term studies of 12-month-old Tg mice, we found
that the microglia-activating properties of NCX-2216 (7.5 mg · kg 1 · d1,
s.c.) were present after 2 weeks of treatment. Microglia were not
activated by NCX-2216 in non-Tg mice lacking A deposits, nor were
microglia activated in Tg animals by flurbiprofen (5 mg · kg1 · d1) alone.
These data are consistent with the argument that activated microglia
can clear A deposits. We conclude that the NO-generating component
of NCX-2216 confers biological actions that go beyond those of typical
NSAIDs. In conclusion, NCX-2216 is more efficacious than ibuprofen or
celecoxib in clearing A deposits from the brains of Tg mice,
implying potential benefit in the treatment of Alzheimer's dementia.
Key words:
Alzheimer's disease; microglia; MHC-II; -amyloid; NSAIDs; transgenic mice
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INTRODUCTION |
Although it is clear that microglia
are intimately involved in the inflammatory response of the brain,
especially to the amyloid deposits in Alzheimer's disease (AD)
(Akiyama et al., 2000 ), their role in the pathogenesis of the disease
is less certain. Microglia have been argued to be responsible for
either -amyloid (A) deposition (Frackowiak et al., 1992; Wegiel
et al., 1996) or A clearance (Schenk et al., 1999; Bacskai et al.,
2000; Bard et al., 2000; Chung et al., 1999; Wyss-Coray et al., 2001).
Microglia are normally kept in a tightly regulated inactive state;
activation is typically short-lived and protective toward neurons and
other neural components (Kreutzberg, 1996; Moore and Thanos, 1996;
Minghetti et al., 1999). However, microglia are chronically active in
late-stage AD. Activated microglia can, in their response to insult,
release reactive oxygen and nitrogen species, with one major
consequence being damage to the local cellular environment (Rogers et
al., 1996; McGeer and McGeer, 1998). The activation of microglia is
probably a response to the deposition of fibrillar A in AD, however
the activation is not a necessary correlate of A deposition. Most
autopsy series involving AD patients and nondemented elderly
individuals demonstrate cases with the classic pathological hallmarks
of AD but without signs of dementia before death (Crystal et al., 1988;
Snowdon, 1997). These individuals also fail to exhibit microglial
reactivity to the A deposits (Lue et al., 1996; Sasaki et al.,
1997).
In recent years, it has been reported that chronic nonsteroidal
anti-inflammatory drug (NSAID) use is able to delay the onset and
possibly reduce the risk of AD (Breitner, 1996; Mackenzie, 1996;
Mackenzie et al., 1998). Although the NSAID data are encouraging, recommendations for chronic NSAID therapy in healthy elderly
individuals are tempered by the well documented risk of
gastrointestinal (GI) bleeding and ulceration (James, 1999). To reduce
the risk of GI injury, new classes of NSAIDs have been developed,
including selective inhibitors of cyclooxygenase-2 (COX2) and nitric
oxide (NO)-donating NSAIDs (nitro-NSAIDs) (Wallace et al., 1994; Del
Soldato et al., 1999). NO plays multiple roles in different
physiological systems, including the brain, where it is potentially a
critical retrograde transmitter (Navarra et al., 2000; Chowdhary and
Townend, 2001; Grassi and Pettorossi, 2001; Napoli and Ignarro, 2001).
The nitro-NSAID used in these studies,
3-[4-(2-fluoro--methyl-[1,1'-biphenyl]-4-acetyloxy)-3-methoxyphenyl]-2-propenoic acid 4-nitrooxy butyl ester (NCX-2216), consists of a nitroxybutyl ester moiety coupled to flurbiprofen via a methoxyphenyl (ferulic acid) linker.
In these studies we used a doubly transgenic (Tg) mouse model of
amyloid deposition (Holcomb et al., 1998) to test the working hypothesis that the benefit of NSAID use in diminishing the risk of AD is attributable to suppression of the microglial response. We compared the effects of a prototypical and nonselective NSAID demonstrated previously to reduce A deposition, ibuprofen (Lim et
al., 2000), with the COX2-selective inhibitor celecoxib and the
NO-releasing NSAID NCX-2216. Unexpectedly, we found that the NO-releasing NSAID activated microglia. In parallel, we also found that
this agent reduced A loads more than the other agents.
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MATERIALS AND METHODS |
Transgenic mice overexpressing Swedish mutant amyloid precursor
protein (APP; Tg2576; K670N/M671L) (Hsiao et al., 1996) and mutant
presenilin-1 (PS1; line 5.1; M146L) (Duff et al., 1996) were crossed to
produce double mutant transgenic APP+PS1 mice. The Alzheimer's-like
phenotype present in these mice has been well characterized (Holcomb et
al., 1998, 1999b; McGowan et al., 1999; Gordon et al., 2001). Chronic
NSAID diet-treated animals were fed drug-containing or control rat chow
between 7 and 12 months of age. Short-term treatments used 12-month-old
mice; the mice were injected daily (subcutaneously) for 14 d with
19:1 castor oil:DMSO vehicle, 7.5 mg · kg1 · d1
NCX-2216, or 5 mg · kg1 · d1
flurbiprofen based on previous work in rats (Hauss-Wegrzyniak et al.,
1999a).
In the long-term administration study, we selected the dosage of
ibuprofen (375 ppm in diet or ~62.5
mg · kg1 · d1
per animal; n = 7 transgenic and 8 nontransgenic mice)
that Lim et al. (2000) found to be effective at reducing A.
We estimated an equivalent dosage of celecoxib (175 ppm or ~30
mg · kg1 · d1
per animal; n = 7 transgenic and 7 nontransgenic mice)
based on the relative human daily dosages of celecoxib and ibuprofen. We similarly estimated an equivalent dosage of NCX-2216 (375 ppm in
diet or ~62.5
mg · kg1 · d1
per animal; n = 7 transgenic mice) so that the ratio of
the mass of flurbiprofen consumed relative to ibuprofen in the first
diet would approximate the relative human dosages of these drugs. We recognize that short of actually measuring the degree of mouse brain
COX inhibition over a 24 hr period, we cannot claim that the dosage of
each drug is equivalent with respect to COX inhibition. However, we
hoped that by administering mass ratios similar to those typically
administered to humans, we would achieve relative brain levels that
were comparable with those typically found in humans using these
medications. Animals were given food and water ad libitum
and kept on a 12 hr light/dark cycle.
Nicox, S.A. (Sophia-Antipolis, France) generously donated NCX-2216. We
purchased ibuprofen from Sigma (St. Louis, MO). For celecoxib,
we used Celebrex capsules (GD Searle & Co., Chicago, IL). All
agents were compounded with rodent chow into pellets (base diet equaled
a modified NIH-31 diet; Harlan, Bicester, UK). Food was kept
refrigerated and placed in the food hoppers every other day. Dosages
are based on a the typical consumption rate of 5 gm/d per mouse (thus
the numbers given above are approximate). Antibodies came from multiple
sources: A was provided by Dr. Paul Gottschall (University of South
Florida), CD-11 (complement receptor-3; antibody Mac-1) rat anti-mouse
monoclonal IgG was obtained from Serotec (Oxford, UK), and major
histocompatibility complex (MHC)-II rat anti-mouse
monoclonal IgG was provided by PharMingen (San Diego, CA). Secondary
antibodies were biotinylated anti-rat IgG and biotinylated goat
anti-rabbit IgG and were visualized using a Vectastain avidin-biotin
complex kit (all from Vector Laboratories, Burlingame, CA). All
other compounds were from Sigma. Reagent grade type I water was
used in all procedures.
Within 24 hr of the last drug administration, animals were overdosed
with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with
25 ml of normal (0.9%) saline. The brains were rapidly removed and
bisected sagittally; the left hemisphere was immersed in freshly depolymerized 4% paraformaldehyde for 24 hr (right hemispheres were
dissected and frozen). The left hemispheres were then cryoprotected by
successive 24 hr immersions in 10, 20, and 30% sucrose in Sorenson's phosphate buffer immediately before sectioning. Fixed, cryoprotected brains were frozen and sectioned in toto in the horizontal
plane at 25 µm using a sliding microtome, collected serially, and
stored at 4°C in Dulbecco's PBS with sodium azide for
subsequent immunohistochemistry and histology. Systematic uniform
random sets of sections with 300 µm spacing were used for all
staining. Sets were stained histologically for Nissl substance using
0.05% cresyl violet and for amyloid deposits by the Congo red
method. Immunohistochemistry was performed on floating sections
using specific antibodies to identify cellular and noncellular markers.
A deposits, both fibrillar and diffuse, were visualized using a
panspecific rabbit polyclonal antibody raised against the
A1-40 peptide. Microglia were visualized with
two antibodies: anti-CR3 and anti-MHC-II. All immunohistochemistry was
visualized with avidin-biotin-horseradish peroxidase and DAB with
nickel enhancement for color development (except for A staining, which omitted the nickel enhancement). Sections stained for microglial markers were secondarily stained with Congo red to localize amyloid deposits in a -sheet configuration. Representative sections from each genotype/treatment group were assayed with the primary antibody omitted to demonstrate nonspecific reaction product formation; these sections displayed negligible staining.
Two regions were examined quantitatively in this experiment: the
hippocampus and the frontal cortex. The hippocampus was delimited by
the corpus callosum laterally and posteriorly, the ventricle and
thalamus medially, and the fimbria/fornix anteriorly. The frontal
cortex is defined as all cortical tissue medial and anterior to the
most anterior limit of the corpus callosum on each hemisection, and
ventral to the initial appearance of the hippocampal formation in the
section set. This region is limited medially by the midline.
The unbiased sampling methods of West et al. (1991) were used to select
sections for quantitative analysis. Because the measured outcome of
total volume occupied by congophilic material and by A
immunocytochemical (ICC) reaction product (ICC-RP) has digital properties (i.e., positive/negative staining readily discriminable by
visual inspection), these stains were quantified using point-counting methods, assisted by a computer-based system, the Stereologer (Systems
Planning and Analysis, Alexandria, VA). Briefly, the equipment
consisted of an X-Y-Z motorized stage (MS-2000; Applied Scientific
Instrumentation, Eugene, OR) and color video camera connected to
a microscope (Olympus BH-2; Olympus Optical, Tokyo, Japan). The digital
output of the camera was processed and displayed by a Macintosh
PowerMac 8500/120AV computer (Apple Computers, Cupertino, CA). The
Stereologer software controlled operation of the system. Total
microglial presence (total immunoreactivity of CR3 ICC-RP) was
quantified using the videodensitometric procedures detailed by Gordon
et al. (1997). This method uses the V150 image analysis system (Oncor,
San Diego, CA) to segment immunostained pixels using
hue-saturation-intensity (HSI) characteristics. Total immunoreactivity is the area occupied by immunopositive pixels multiplied by the optical density of those pixels. Because the number
of MHC-II-positive microglial profiles in vehicle-treated mice was too
low to be reliably estimated using a reasonable number of disectors,
identifiable profiles in all animals were manually counted and are
reported as average number of MHC-II-positive profiles per section.
Between 10 and 15 sections per region per animal were examined for each measure.
At necropsy, gastrointestinal tracts (stomach, small intestine, and
large intestine) of all mice were collected and post-fixed for 24 hr in
4% paraformaldehyde. The major portion of the gastrointestinal tract
was embedded in paraffin, sectioned, and stained with hematoxylin and
eosin. These sections were independently evaluated by three investigators who were unaware of the treatment conditions.
To assess possible treatment-related differences in A deposition and
microglial number and total immunoreactivity, the measurements for each
brain region of each subject were analyzed by ANOVA, followed by least
significant difference post hoc analyses using the
computer program Statview (SAS Institute, Cary, NC).
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RESULTS |
The anterior cortex and hippocampus of APP+PS1 and nontransgenic
mice were examined after 5 months of ad libitum
administration of NCX-2216, ibuprofen, celecoxib, or the base diet. As
expected, the transgenic mice accumulated considerable A in the
cerebral cortex and hippocampus as they reached 12 months of age (Fig. 1 A,C). Mice treated with
NCX-2216 between 7 and 12 months of age had a reduction in the amounts
of amyloid found in these structures (Fig. 1B,D).
When quantified by stereology, there was a significant effect of drug
treatment in the cerebral cortex (ANOVA;
F(3,21) = 5.7; p < 0.005) and hippocampus (F(3,21) = 5.9;
p < 0.005). The greatest
reduction was found in the mice treated with NCX-2216; these mice
displayed 40-45% less A load than mice given control diets
(p < 0.001) (Fig.
2A,B).
Ibuprofen also reduced the A load by 20-25%, with a significant
reduction in the cerebral cortex (p < 0.025)
(Fig. 2A). The mean values for celecoxib-treated mice were 15-20% lower than the mean for control animals, but there was no
significant difference with the statistical power of the present study
(Fig. 2A,B).
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Figure 1.
A immunohistochemistry in control and
NCX-2216-treated transgenic mice. A immunohistochemistry of the
frontal cortex (A) and hippocampus
(C) of an APP+PS1 transgenic mouse fed the
control diet from 7 to 12 months of age is shown. A
immunohistochemistry of the frontal cortex (B)
and hippocampus (D) of an APP+PS1 transgenic
mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also
shown. Scale bar, 1 mm.
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Figure 2.
A load in the anterior cortex and hippocampus
of NCX-2216-treated mice. The A load is the percentage of the
cross-sectional area occupied by the immunohistochemical reaction
product. NCX-2216-treated APP+PS1 mice have significantly less A in
both the cortex (A) and hippocampus
(B), as demonstrated by immunohistochemistry,
compared with mice treated with ibuprofen (hippocampus,
p < 0.05), celecoxib (cortex,
p < 0.05; hippocampus, p < 0.005), or fed the control diet (cortex, p < 0.001; hippocampus, p < 0.001). In the cortex,
ibuprofen significantly reduced A load as well
(p < 0.025) compared with mice fed the
control diet.
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A similar result was observed for the Congo red-stained sections,
although the reductions are restricted to the NCX-2216-treated mice.
The numbers of congophilic deposits in the cerebral cortex and
hippocampus were reduced in the mice fed NCX-2216 between 7 and 12 months of age, and the deposits that were present appear smaller (Fig.
3). ANOVA revealed a significant effect
of drug treatment on the Congo red-stained area in the cerebral cortex (F(3,21) = 6.7; p < 0.005) (Fig. 4A) and in
the hippocampus (F(3,21) = 4.7;
p < 0.01) (Fig. 4B). Mean
comparisons indicated reductions of 35-40% in both regions for the
mice treated with NCX-2216 (p < 0.01) (Fig.
4A,B). In fact, the NCX-2216-treated mice had
significantly lower Congo red staining than the
other NSAID-treated groups in both
structures (p < 0.02).
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Figure 3.
Congo red staining in NCX-2216-treated transgenic
mice. Shown is Congo red staining of the frontal cortex
(A) and hippocampus (C) of
an APP+PS1 transgenic mouse fed the control diet from 7 to 12 months of
age. Congo red staining of the frontal cortex (B)
and hippocampus (D) of an APP+PS1 transgenic
mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also
shown. Scale bar, 1 mm.
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Figure 4.
Amyloid load in the anterior cortex and
hippocampus of NCX-2216-treated mice. Amyloid load is the percentage of
the cross-sectional area occupied by Congo red stain. NCX-2216-treated
APP+PS1 mice have significantly less amyloid in both the cortex
(A) and hippocampus (B), as
demonstrated by Congo red staining, compared with mice treated with
ibuprofen (cortex, p < 0.002; hippocampus,
p < 0.005), celecoxib (cortex,
p < 0.02; hippocampus, p < 0.02), or fed the control diet (cortex, p < 0.001;
hippocampus, p < 0.01).
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The most surprising result of this treatment was the effect of the
drugs on microglial activation. NCX-2216 treatment dramatically increased the numbers of activated microglia that are stained with
MHC-II antisera over the small numbers of cells visible in control
transgenic mice (there were no detectable MHC-II-stained cells in
one-half of the control mice) (Fig.
5A,C). For the most part,
these positively stained microglia were in association with congophilic
amyloid deposits and absent in regions lacking such deposits. In the
anterior cortex of mice treated with NCX-2216, there was a significant
increase in MHC-II-positive cells over mice fed control diets
(p < 0.05) (Fig.
6A). In the
hippocampus, mice treated with NCX-2216 had more
MHC-II-immunopositive profiles than all three other groups
(p < 0.02) (Fig. 6B). In both
structures, ibuprofen and celecoxib treatment resulted in a small but
nonsignificant increase in the number of these cells compared with mice
on control diets (Fig. 6).
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Figure 5.
MHC-II immunohistochemistry with Congo red
counterstaining of NCX-2216-treated transgenic mice is shown. MHC-II
immunohistochemistry (black reaction product) of the
frontal cortex (A) and hippocampus
(C) of an APP+PS1 transgenic mouse fed the
control diet from 7 to 12 months of age is shown. MHC-II
immunohistochemistry of the frontal cortex (B)
and hippocampus (D) of an APP+PS1 transgenic
mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also
shown. Scale bar, 100 µm.
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Figure 6.
NCX-2216-treated APP+PS1 mice have larger numbers
of MHC-II-positive microglia in the frontal cortex
(A) and hippocampus (B)
compared with mice fed a control diet (frontal cortex,
p < 0.05; hippocampus, p < 0.02). Ibuprofen-treated mice have a small but not significant increase
in the numbers of MHC-II-positive microglia in the anterior cortex
compared with control-treated mice.
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We have also demonstrated that microglial expression of MHC-II can be
elicited after shorter administrations of NCX-2216. Subcutaneous
injection of 7.5 mg · kg1 · d1
NCX-2216 for 2 weeks increased the numbers of MHC-II-positive microglia
in APP+PS1 transgenic mice at 12 months of age (Figs. 7A,B,
8A). Immunostaining for
another microglial marker, CD11 (complement receptor-3 stained with the
Mac-1 antibody), also appeared to be modified by the NCX-2216 treatment
(Fig. 7C,D). However, in this case the main effect was
greater staining intensity rather than increased numbers of stained
cells (microglia express CD11 even when quiescent). To quantify this
effect, we established an intensity threshold for the HSI segmentation
that would not include resting microglia in the segmentation window. We
then measured the area occupied by this intense reaction product (using a constant segmentation threshold for each section as described by
Gordon et al., 1997), and found an increased staining area in the
hippocampus of mice treated with NCX-2216 compared with control mice
(Fig. 8B) (p < 0.05). Not
surprisingly, there were no reductions in A load or Congo red
staining after this short treatment period (data not shown).
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Figure 7.
Effects of short-term NCX-2216 treatment on
microglial markers in APP+PS1 mice. Animals were treated with 7.5 mg · kg1 · d1
NCX-2216 or vehicle for 14 d before they were killed.
MHC-II immunohistochemistry of the frontal cortex of an APP+PS1
transgenic mouse treated with vehicle (A) and
NCX-2216 (B) is shown. CD-11 immunohistochemistry
of the frontal cortex of an APP+PS1 transgenic mouse treated with
vehicle (C) and NCX-2216
(D) is also shown. Scale bar, 100 µm.
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Figure 8.
Measurement of the effects of short-term
NCX-2216 treatment on microglial markers in APP+PS1 mice. Animals were
treated with 7.5 mg · kg1 · d1
NCX-2216 or vehicle for 14 d before they were killed. Sections
were stained with anti-MHC-II antibodies and Congo red, and positive
microglial profiles were counted on systematic uniform random sections
through the frontal cortex and hippocampus. Results are expressed as
average number of MHC-II-positive microglial profiles per section in
each region (A). NCX-2216 treatment resulted in a
>10-fold increase in the average number of MHC-II-positive profiles in
both the frontal cortex and hippocampus when compared with
vehicle-treated mice (p < 0.025). Sections
were stained with anti-CR3 antibody and Congo red, and total
immunoreactivity measurements of ICC-RP were calculated
(B). There is significantly greater total
immunoreactivity of Congo red-associated ICC-RP in the hippocampus
(p < 0.05); however, there is only a trend
toward greater total immunoreactivity in the frontal cortex
(p < 0.2).
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To understand the mechanism of this activation better, we examined
APP-only transgenic mice at 12 months of age. Although these mice have
considerably fewer A deposits, there was still a significant effect
of NCX-2216 on the numbers of MHC-II-positive profiles (Fig.
9). This property of activating microglia
is apparently not solely a function of the NSAID moiety of the
molecule, because injections of flurbiprofen alone had no effect on the
numbers of MHC-II-stained microglia (Fig. 9). Importantly, the
activation of microglia by NCX-2216 involves an interaction with the AD
phenotype of the mice. MHC-II-positive microglia were not found in
nontransgenic mice treated with either NCX-2216 or vehicle (Fig.
9).
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Figure 9.
Measurement of MHC-II immunohistochemistry after
acute treatment of APP transgenic and nontransgenic mice with NCX-2216,
flurbiprofen, or vehicle. NCX-2216 (7.5 mg · kg1 · d1)
treatment of 12-month-old APP transgenic mice results in a
significantly increased number of MHC-II-positive microglia compared
with APP mice treated with either flurbiprofen (5 mg · kg1 · d1) or
vehicle (p < 0.001). Nontransgenic mice
treated daily with either NCX-2216 or vehicle for 2 weeks demonstrate
no detectable MHC-II-positive microglia.
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Histological examination of gastrointestinal tract sections in mice
treated with NSAIDs for 5 months found degrees of erosions, gastritis,
and ulcers in each treatment condition. Gastrointestinal erosions were
found in 6 of 12 control mice, 4 of 13 mice treated with celecoxib, 1 of 7 mice administered NCX-2216, and 6 of 15 mice given ibuprofen.
Gastritis was found in two of the celecoxib-treated mice, one
NCX-2216-treated mouse, and five of the ibuprofen-treated mice. Ulcers
were found in two of the NCX-2216 mice. Two ibuprofen-treated mice died
during the course of the study, but no deaths were found in the other
groups. None of the treatment groups lost weight over the course of the
study, and there was no statistically significant difference in weight
gain between the groups during the course of the study. In other
studies, the parent of NCX-2216, flurbiprofen, led to severe
gastropathy, weight loss, and in some instances death when administered
to rats or mice for 2 weeks, indicating that the histopathology
observed here was relatively benign (Wallace et al., 1994) (J. Wallace,
unpublished observations).
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DISCUSSION |
The major finding of this study is that the nitric oxide-releasing
NSAID NCX-2216 substantially reduced A loads in APP+PS1 transgenic
mice. Ibuprofen also had an effect on diffuse A, confirming the
original findings of Lim et al. (2000) in transgenic mice that
overexpress only the APP transgene. Although NCX-2216 does not
completely clear brain amyloid, it should be noted that level of A
production in these doubly transgenic mice is 5- to 15-fold higher than
in mice expressing APP alone (Morgan et al., 2000). This
amyloid-reducing effect of NCX-2216 is greater than that obtained with
vaccination in the same mouse model (Morgan et al., 2000). To date,
NCX-2216 is the most efficacious agent we have found in reducing A loads.
We also observed activation of microglia in the immediate vicinity of
fibrillar A deposits by NCX-2216. This was evidenced by induction of
MHC-II expression and increased staining for complement receptor-3. It
should be noted that this increase in microglial activation occurred
despite reduced Congo red-stained amyloid deposits (in chronically
treated mice), which act as a stimulus for microglial activation as
transgenic mice age (Masliah et al., 1996; Frautschy et al., 1998;
Benzing et al., 1999; Stalder et al., 1999; Matsuoka et al., 2001).
This outcome was in opposition to our original assumption that
anti-inflammatory drugs would decrease the microglial activation caused
by A deposition.
The inflammation hypothesis of AD posits that hyperactive microglia are
the proximal cause of AD-associated neurotoxicity and contribute
directly to the neuropathology characteristic of the disease (McGeer
and McGeer, 1998; Akiyama et al., 2000). A number of reports
show that chronic NSAID use is associated with reduced risk for
dementia (Breitner, 2000) and may even slow progression of the outward
symptoms of AD (Rogers et al., 1993). At autopsy, a significant
fraction of cognitively normal aged individuals present
densities of amyloid plaques and neurofibrillary tangles consistent
with a diagnosis of AD (Crystal et al., 1988; Davis et al., 1999).
Mackenzie and Munoz (1998) report that microglial reactivity associated
with amyloid pathology is reduced in cognitively normal arthritics
taking anti-inflammatory compounds, suggesting that NSAIDs might reduce
the risk of AD by inhibiting microglial activation. However, Lue et al.
(1996, 2000) generally found low microglial activation in these
"high-plaque normal" cases, regardless of NSAID use. Halliday et
al. (2000) failed to find reduced microglia reactivity in demented
individuals taking NSAIDs.
In addition to the potential role of microglial activation causing
damage in AD, there is increasing evidence, at least in transgenic
models of amyloid deposition, that activated microglia may remove
amyloid deposits. In vitro, it is clear that microglia are
capable of phagocytosing A aggregates via scavenger A or receptors
for advanced glycosylation endproducts (Shaffer et al., 1995;
Ard et al., 1996; Paresce et al., 1996; Webster et al., 2000). In the
presence of anti-A antibodies, Fc receptors may also mediate amyloid
uptake in vitro or in situ (Bard et al., 2000;
Brazil et al., 2000; Webster et al., 2001). Injections of A amyloid
into rat brain also elicit a phagocytic response by microglia, leading
to removal of large fractions of the injected material (Frautschy et
al., 1992; Weldon et al., 1998; Holcomb et al., 1999a). In transgenic
mice, activation of microglia has been argued to be responsible for the
reductions in A deposition found with vaccination (Schenk et al.,
1999; Wilcock et al., 2001) or with direct injection of anti-A
antibodies (Bacskai et al., 2000); however, a peripheral action of
antibodies in removing A from brain has also been proposed (DeMattos
et al., 2001). Furthermore, lipopolysaccharide (LPS) administration,
which also activates microglia, results in reductions in A
deposition (DiCarlo et al., 2001). These data suggest that the
increased microglia activation may have caused the reduced levels of
amyloid in the transgenic mice treated with NCX-2216 by phagocytosis
and clearance of the A deposits.
The mechanism by which NCX-2216 leads to microglial activation is not
clear. There are three potentially active components of the NCX-2216
molecule. The first is the NSAID moiety, flurbiprofen. The majority of
research on the actions of NSAID on neural cells has been conducted
using cells activated by chemical irritants or the endotoxin LPS. Under
these circumstances, NSAIDs reduce the inflammatory response. In young
rats infused with LPS, even NCX-2216 acts like a typical NSAID and
reduces microglia activation (Hauss-Wegrzyniak et al., 1999a,b).
However, in naive mice, Prechel et al. (2000) reported the upregulation
of a microglial marker, complement receptor-3, with indomethacin.
Studies outside of the CNS have found that the COX product
prostaglandin E2 (PGE2) normally maintains cells of monocytic origin, such as microglia, in a resting condition. Inhibition of constitutive COX activity by NSAIDs reduces PGE2 and disinhibits monocytes, resulting in
activation of these cells (Young, 1994; Zicari et al., 1995). Indeed,
in old rats, NCX-2216 increased the numbers of activated microglia in
the presence of an LPS dose that had little effect of its own
(Hauss-Wegrzyniak et al., 1999a). It is possible that under certain
circumstances, high brain levels of NSAIDs may activate microglia, as
observed here (note that ibuprofen and celecoxib tended toward
increased MHC-II staining as well). Little is known regarding the
effects of the modifications present in NCX-2216 on the distribution of the drug into various tissues, particularly the brain. If one action is
to enhance blood-brain-barrier penetration, this might explain the
greater effects of NCX-2216 versus flurbiprofen alone. This issue is
being addressed in ongoing studies.
The second component of the NCX-2216 molecule is ferulic acid, an
antioxidant found in a number of fruits and vegetables (Graf, 1992). It
is also used commercially to retard food spoilage. Yan et al. (2001)
reported recently that pretreatment of mice with ferulic acid protected
these mice from learning and memory deficits caused by intraventricular
injections of A. Interestingly, these authors also found that the
treatment of naive mice with ferulic acid could elevate GFAP and
interleukin-1 (IL-1) levels, at least transiently.
A third possibility is that the slow generation of nitric oxide by
NCX-2216 is conferring novel properties to the NSAID. In multiple
instances, NO-releasing side chains attached to parent molecules result
in more potent actions than the parent compound, or confer additional
actions not found with the parent compound alone. For example, addition
of an NO-releasing group to acetaminophen (a.k.a. paracetamol) adds
anti-inflammatory activity to this compound that is typically useful
only for antipyretic and analgesic actions. The nitroacetaminophen even
avoids the liver toxicity caused by high doses of acetaminophen alone
(al Swayeh et al., 2000; Futter et al., 2001). Addition of an
NO-releasing group to aspirin provides greater protection against
restenosis and atherosclerosis in models of cardiovascular disease
(Napoli et al., 2001) and, unlike aspirin alone, directly blocks IL-1
induction by inhibiting IL-1-converting enzyme and other
caspases (Fiorucci et al., 2000). The inability of flurbiprofen to
mimic the microglial activating properties of low-dose NCX-2216 in the
short-term treatment studies implies some unique actions of the
modified NSAID. This may result from the presence of ferulic acid, the
NO-generating group, or both, in conjunction with the NSAID moiety.
Future experiments will address this issue in more detail.
Given the multiple pathways for microglia activation in the brain and
the complex interplay of stimuli present in the AD brain, it is likely
that there are multiple states of microglial activation. As suggested
by Raine (2000), it is plausible that the activation of immune system
components in the AD brain may have benefits rather than exclusively
detrimental effects. Roher and colleagues (Roher et al.,
1993a,b; Kuo et al., 1998) have demonstrated that in end-stage
Alzheimer's disease, the A deposits have been chemically modified
by racemization, isomerization, glycation, truncation, transglutamination, etc. These "hard" deposits may be difficult for
microglia to clear from the tissue, leading to a condition of
"frustrated phagocytosis" and induction of extreme activation states involving respiratory burst activity, among other potentially destructive reactions. This condition has been referred to by McGeer
and McGeer (2001) as "autotoxicity" to distinguish it from other
forms of glial activation. Importantly, it is likely that these extreme
forms of microglial activation might be moderated by NSAIDs or other
anti-inflammatory agents, while leaving constitutive phagocytic
functions intact. In this context, a potential mechanism of NSAID
protection from AD may involve an early "release" of microglia from
their resting state, before the hardening of the amyloid deposits,
permitting effective phagocytic removal of A at an early stage of
deposition. Furthermore, NSAIDs have been shown to inhibit the
expression of a wide variety of inflammatory mediators that have been
suggested as potential pathological chaperones of A deposition, such
as the interleukins and tumor necrosis factors. By this mechanism
alone, NSAIDs may be able to reduce A deposition. This reduced A
load would decrease the likelihood of a more severe inflammatory cycle
being initiated later in the disease process, when the deposits are
more difficult to clear effectively. Importantly, the "soft" A
deposits of transgenic mice do not have the same degree of modification
as those found in the end-stage AD brain (Kuo et al., 2001) This
hypothesis would also predict that NSAID use years before the expected
onset of dementia would have the greatest benefit in protecting from
AD. From previous work we have published on spatial memory deficits in
APP+PS1 mice (Morgan et al., 2000; Gordon et al., 2001), we anticipated
that at 12 months of age, the transgenic mice given control diets would
not be old enough to exhibit reliable learning deficits. Thus, we did
not test this group of animals. We have initiated a second study in
which we will treat mice to an age at which behavioral deficits are
apparent in untreated transgenic mice, and expect to test them for
spatial learning in the near future.
In conclusion, these data identify an unexpected activation of
microglia by a nitro-NSAID in a mouse model of amyloid deposition. These results may impact the design of human trials to evaluate these
drugs as therapeutics in AD patients. They also point to the
possibility that some microglial activation in AD might be considered
beneficial, when in the absence of stimulation of autotoxic reactions.
We plan to extend these findings by comparing NCX-2216 and its
component parts in additional long-term studies examining the
functional, behavioral, and histopathological consequences of such
treatments in these transgenic models of amyloid deposition.
| |
FOOTNOTES |
Received Sept. 10, 2001; revised Dec. 4, 2001; accepted Dec. 17, 2001.
This work was supported by National Institutes of Health Grant AG 15490 (M.N.G.) and the Benjamin Trust (D.M.). We thank Piero del Soldato of
Nicox, S.A. for the generous donation of NCX-2216 and Ennio Ongini for
his thoughtful comments on this work.
Correspondence should be addressed to Dave Morgan, University
of South Florida College of Medicine, MDC Box B9, Tampa, FL 33612. E-mail: dmorgan{at}hsc.usf.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
