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A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland; and
Department of Clinical Pathology, Kuopio University Hospital, Kuopio, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-converting enzyme and
inducible nitric oxide synthase up-regulation in animal models of
ischemic stroke and Huntington’s disease and is therapeutic in these
disease animal models. Here we report that nanomolar concentrations of
minocycline protect neurons in mixed spinal cord cultures against NMDA
excitotoxicity. NMDA treatment alone induced microglial proliferation,
which preceded neuronal death, and administration of extra microglial
cells on top of these cultures enhanced the NMDA neurotoxicity.
Minocycline inhibited all these responses to NMDA. Minocycline also
prevented the NMDA-induced proliferation of microglial cells and the
increased release of IL-1 and nitric oxide in pure microglia
cultures. Finally, minocycline inhibited the NMDA-induced activation of
p38 mitogen-activated protein kinase (MAPK) in microglial cells, and a
specific p38 MAPK inhibitor, but not a p44/42 MAPK inhibitor, reduced
the NMDA toxicity. Together, these results suggest that microglial
activation contributes to NMDA excitotoxicity and that minocycline, a
tetracycline derivative, represents a potential therapeutic agent for
brain diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have suggested that inflammation, involving nonneuronal
cells such as infiltrating leukocytes and parenchymal microglia,
contributes to the delayed enlargement of ischemic brain injury
(1, 2, 8), amyloid-induced neurotoxicity in Alzheimer’s
disease (9, 10), and demyelination in multiple sclerosis
(11, 12). The key players in inflammation are glial cells,
both astrocytes and microglia, which are activated in these brain
diseases and in response to glutamate excitotoxicity
(13, 14, 15). Activated microglia release a large variety of
neurotoxins, including the free radicals hydrogen peroxide, superoxide,
and NO, that also is able to nitrate proteins; glutamate and
quinolinic acid; extracellular proteases; eicosanoids; and cytokines,
such as TNF-
and IL-1, which further increase microglial
proliferation and activation, thereby increasing the release of
microglial toxins (8, 13, 14, 16, 17). Importantly,
several studies have shown that the neurotoxicity of microglial toxins
is mediated through the NMDA receptor (NMDAR), involving either
glutamate and quinolinic acid or a still uncharacterized NMDAR binding
molecule (18, 19, 20, 21). Because inflammation is a delayed and
prolonged response to brain injury, it is regarded as a tempting
pharmacological target with a potentially wide therapeutic window,
unlike NMDAR antagonists that must be delivered immediately after a
brain insult and have strong side effects (2, 22, 23).
We have previously shown that minocycline, a semisynthetic tetracycline
derivative, is able to provide neuroprotection against global ischemia
in gerbils and focal brain ischemia in rats (24, 25). In
addition, minocycline delays mortality in a transgenic mouse model of
Huntington’s disease (26). In all these studies, the
protective effect of minocycline was associated with reduced activation
of inducible NOS and IL-1-converting enzyme, which are mainly
expressed by microglia. In addition, ischemia-induced proliferation of
microglia was inhibited by minocycline. In the present study, we show
that NMDA-induced neuronal death involves proliferation and activation
of microglial cells and that minocycline prevents completely the NMDA
toxicity and the preceding activation and proliferation of microglial
cells. These results support the notion that microglial activation
contributes to excitotoxic neuronal death, which can be inhibited by
antiinflammatory compounds, such as minocycline.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SC were dissected out from 14-day-old Wistar rat embryos
(University of Kuopio, Finland), and the meninges and dorsal root
ganglia were removed. Tissues were minced and trypsinized (0.25%
trypsin-EDTA in 0.1 M phosphate buffer; Life Technologies, Roskilde,
Denmark) for 15 min at 37°C. After centrifugation for 5 min at 800
rpm, the tissues were resuspended into DMEM (high glucose, containing
200 µg/ml CaCl, 97.67 µg/ml MgSO4, and 30 µg/ml
glycine; Life Technologies) containing 10% FBS and 10%
heat-inactivated horse serum (HS-HI), and triturated with a
fire-polished Pasteur pipette. A single-cell suspension was collected,
and the cell density of the suspension was counted with a Burker
hemocytometer. Cells were cultured onto
poly-L-lysine-coated 96-well plates (1 x
105 cells/well) or 24-well plates (2.5 x
105 cells/well) and maintained at 37°C in a 7.5%
CO2 incubator. The medium was changed on the following day
to DMEM containing 5% FBS and 5% HS-HI. After 4 days in vitro, 5 µM
cytosine -D-arabinofuranosylcytosine (Sigma, St. Louis,
MO) was added for 24 h to inhibit the growth of nonneuronal cells.
This procedure results in mixed SC cultures, consisting of all neuronal
populations (70%) present in the SC, astrocytes (25%), and few other
nonneuronal cell types, including microglia (5%). We have previously
shown that these neurons express functional glutamate receptors
(27).
Primary mixed glial and microglia cultures
Cortices and midbrains were dissected out from newborn Wistar
rat puppies, and the meninges and blood vessels were removed. Tissues
were collected into 0.1 M PBS and washed four times with cold 0.1 M
PBS, homogenized mechanically without enzymes, and filtered through a
70-µm pore size nylon cell strainer (Falcon; Fisher Scientific,
Pittsburgh, PA). PBS was removed by two centrifugation steps (1000
x g for 10 min at 4°C) and replaced with DMEM (low
glucose, containing 200 µg/ml CaCl, 97.67 µg/ml MgSO4,
and 30 µg/ml glycine; Life Technologies) + 10% FBS. The cells
were suspended into culture medium and plated onto cell culture plates
at a density of 1 x 105 cells/mm2. The
cultures were maintained in a humidified incubator at 37°C and 5%
CO2. The medium was changed at the second day in vitro and
once a week thereafter. This procedure results in mixed glial cultures,
consisting of dividing astrocytes and microglial cells. After 2 weeks
in vitro, microglia was harvested once a week by carefully collecting
the medium without shaking until the mixed cultures were 2–3 mo old.
The purity of the microglial cultures was tested by immunocytochemical
staining for OX-42, a microglia marker, and for glial fibrillary acidic
protein, an astrocyte marker. Also, the cellular morphology was
carefully investigated under phase contrast microscope. The purity of
microglial cultures was found to be 
98%.
Cell exposure experiments
SC cultures were exposed at seventh day in vitro to 300 µM NMDA (Research Biochemical International, Natick, MA) for 5 min and analyzed 24 h after the onset of exposure. This NMDA treatment reduces the number of surviving neurons to 40–50%. To some cultures 20 nM minocycline (Sigma), 10 µM PD98059 (Tocris Cookson, Bristol, U.K.), a specific p44/42 mitogen-activated protein kinase (MAPK) inhibitor, or 10 µM SB203580 (Tocris Cookson), a specific p38 MAPK inhibitor, was administered 30 min before the NMDA treatment. All of the compounds were dissolved in the culture medium supplemented with 5% HS-HI, which was used alone as a 0 control. The cultures were analyzed 24 h after the onset of exposures, unless mentioned otherwise. In a set of experiments, microglial cells were cultured on top of 5-day-old SC cultures and exposed at day 7 as described above. Mixed glial cultures were exposed to 500 µM NMDA after 2 wk in vitro, and pure microglial cultures were exposed on the third day in vitro. Minocycline (200 nM) or 10 µM MK-801 (Tocris Cookson), a specific NMDAR antagonist, was administered to some cultures 30 min before exposure. All compounds were dissolved in the cell culture medium supplemented with 10% FBS, which was used alone as a 0 control.
Lactate dehydrogenase (LDH) assay
The release of LDH was measured from the culture medium using a Sigma Kinetic LDH kit. The culture medium samples were collected 24 h after the onset of excitotoxic exposures (unless mentioned otherwise), prepared cell free by centrifugation, and measured immediately with a Multiskan MS ELISA reader (Labsystems, Helsinki, Finland) taking seven absorbance measurements during 3 min at 30-s intervals at a wavelength of 340 nm.
NO and IL-1 assays
The production of NO was measured by measuring the released NO
metabolites (nitrates and nitrites) with Griess reagent (Sigma). After
a 24-h exposure, the culture medium samples were collected and prepared
cell free by centrifugation. Fifty microliters of the medium were
incubated with the same volume of Griess reagent at room temperature
for 15 min before measuring OD540 in a Multiskan
ELISA reader (Labsystems) with appropriate standards. IL-1 samples
were prepared similar to NO samples and determined using a rat IL-1
ELISA kit (Endogen, Woburn, MA) according to the manufacturer’s
instructions and a Multiskan MS ELISA reader (Labsystems).
Immunocytochemistry
The cultures were fixed with 4% paraformaldehyde in 0.1 M PBS for 20–30 min and rinsed in 0.1 M PBS. The nonspecific binding was blocked with 1% BSA and 0.3% Triton X-100 in 0.1 M PBS for 30 min at room temperature. Subsequently, the cultures were incubated with primary Abs to neurons (mouse mAb, against neuronal nuclei; Chemicon, Temecula, CA; 1:100 dilution), to microglia (mouse mAb, OX-42, against CD11b surface Ag; Serotec, Oxford, U.K.; 1/1500 dilution), to phospho-p38 or -p42/44 MAPK (rabbit polyclonal antibody, Phospho-p38 or -p42/44 MAPK; New England Biolabs, Beverly, MA; 1/1000 and 1/250 dilution) in the blocking buffer for 48 h at 4°C. The cultures were rinsed with 0.1 M PBS, incubated with biotinylated anti-mouse IgG (Amersham, Buckinghamshire, U.K.; 1/200 dilution) or biotinylated anti-rabbit IgG (Amersham; 1/200 dilution) secondary Ab for 2 h at room temperature, and reacted with alkaline phosphatase-avidin-biotin complex (Vector Laboratories, Burlingame, CA; 1/200 dilution) for 2 h at room temperature and rinsed. The color was obtained from alkaline phosphatase substrate kit (Vector Laboratories). In a set of experiments, phospho-p38 MAPK-immunostained cultures were double-stained with OX-42 Ab (1/800 dilution) using anti-mouse IgG fluorescein (Jackson ImmunoResearch Laboratories, West Grove, PA; 1/70 dilution) as a secondary Ab. Immunoreactive cells were counted from 6–10 random fields of 4 x 10-3 mm2 area per well and from 3–5 wells per treatment.
Proliferation assay
The thymidine analogue BrdU (Sigma) was added to the medium at 5 µM concentration. After 24 h, the cultures were fixed with 4% paraformaldehyde in 0.1 M PBS for 20–30 min and rinsed in 0.1 M PBS. The DNA was denaturated by incubating the cultures first with 50% formamide in 2x SSC for 2 h at 65°C followed by 2 N HCl treatment for 30 min at 37°C. After neutralization with 0.1 M boric acid, pH 8.5, for 10 min at room temperature, the cultures were rinsed in 0.1 M PBS and incubated with a mouse monoclonal anti-BrdU (0.25 µg/ml anti-BrdU; Boehringer Mannheim, Indianapolis, IN) in the blocking buffer at room temperature for 24 h. Otherwise, the procedure was identical with the immunocytochemistry described above. The immunoreactive cells were counted in a ratio of total cell number from 6–10 random fields of 4 x 10-3 mm2 area per well and from 3–5 wells per treatment.
Statistical analysis
Data are presented as the mean ± SD. Statistical comparisons were made by single-factor ANOVA followed by Tukey’s post hoc test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 5-min exposure to 300 µM NMDA resulted in increased LDH
release from dying neurons measured 24 h after the onset of
exposure (Fig. 1
A). Treatment
with 20 nM minocycline started 30 min before NMDA exposure was able to
prevent neuronal death. The reduced neuronal loss in
minocycline-treated cultures was further confirmed by counts of
NeuN-stained neurons (Fig. 1B). Cell death was mainly
necrotic in this cell culture model, in that no apoptotic fragmentation
was seen with bisbenzimide staining (data not shown). The NMDA-induced
neuronal death was associated with increased NO metabolite production
(from 1–3 µM to 5–20 µM), which was inhibited by 20 nM
minocycline (Fig. 1C).
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A 5-min exposure to 300 µM NMDA caused a 1.9-fold increase in
the number of OX-42 immunoreactive microglial cells in SC cell cultures
within 24 h (Fig. 2A).
Administration of 20 nM minocycline 30 min before the exposure
prevented the NMDA-triggered increase of microglial cells. Minocycline
even slightly prevented the spontaneous proliferation of microglia.
Even sublethal doses (50 µM for 5 min) of NMDA increased the number
of microglial cells (not shown), suggesting that the microglial
proliferation was not secondarily due to increased neuronal death. When
neuron-free mixed glial cultures were exposed to 500 µM NMDA for
24 h, a 5.2-fold increase of OX-42-positive cells was observed,
and this increase was again reduced close to basal levels by 200 nM
minocycline treatment (Fig. 2B). To investigate whether
microglial proliferation caused by NMDA stimulation is mediated by
other glial cells, pure microglial cultures were exposed to 500 µM
NMDA for 30 min or 24 h in the presence of 5 µM BrdU. BrdU
incorporates into DNA of proliferating cells, which can be detected by
anti-BrdU Ab. NMDA administered for either 30 min or 24 h
caused a 2- to 4-fold increase of the microglial proliferation ratio,
which was prevented by 200 nM minocycline or 10 µM MK-801 (Fig. 2C).
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and NO release in pure
microglial cultures. Twenty-four hours after addition of 500 µM NMDA,
increased levels of IL-1 (from 2–4 µM to 4–10 µM) and NO (from
1–3 µM to 5–20 µM) metabolites were detected (Fig. 3). The release of both IL-1 and NO
metabolites was inhibited by 200 nM minocycline treatment before
exposure. We also confirmed that significant NO release occurred both
after 5 min and 24 h exposure to NMDA and that MK-801 was able to
block the NO release (not shown).
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To study whether NMDA-induced microglial activation and
proliferation precedes or is a consequence of neuronal death, we
compared the time course of the changes in OX-42-immunoreactive cells
and LDH release during a 24-h follow-up period (Fig. 4A). The number of
OX-42-immunoreactive cells was increased significantly as early as
1 h after a 5-min exposure to 300 µM NMDA and was further
increased at later time points. LDH release, on the other hand, was not
significantly increased until 12 and 24 h after NMDA challenge
(Fig. 4A), indicating that induction of CD11b surface Ag and
microglial proliferation starts before neuronal death. To study further
the role of microglia in NMDA excitotoxicity, microglial cells were
harvested and added on top of mixed SC cultures. This procedure
increases the microglia-neuron ratio from 0.071 to 0.185.
Administration of extra microglia on SC cultures increased the
NMDA-triggered LDH release 3-fold, which was significantly, but not
completely, inhibited by minocycline (Fig. 4B).
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Because MAPKs have been reported to be involved in microglial
activation and induction of inducible NOS, we studied whether a 10-min
exposure to NMDA causes changes in activation of p38 and p44/42 MAPKs
in SC cultures. Immunoblots did not reveal detectable levels of p38
MAPK (not shown), suggesting that p38 MAPK may be activated only in a
minority of the cultured cells. By immunocytochemistry, the
phosphorylated form of p38 MAPK was detected only in microglial cells,
which was confirmed by double staining with OX-42 Ab (Fig. 6, A and B). The number of p38 MAPK-positive
microglia was significantly (130%) elevated by NMDA exposure (Fig. 5). Minocycline administration (20 nM) 30
min before the onset of NMDA exposure decreased the number of p38
MAPK-immunoreactive microglial cells to the basal levels (Fig. 6, A–D). The basal levels of
phosphorylated p44/42 MAPK were high in both astrocytes and neurons
(Fig. 6, E–G). NMDA increased the number of p44/42
MAPK-positive neurons by 41.3% (p < 0.005;
ANOVA, Tukey’s post hoc test), but 20 nM minocycline treatment reduced
the number of immunoreactive neurons by 40% (not shown). To dissociate
the role of p38 MAPK activation in microglial cells and p44/42 MAPK in
astrocytes and neurons, 10 µM SB203580, a specific p38 MAPK
inhibitor, or 10 µM PD98059, a specific p44/42 MAPK inhibitor, was
administered to mixed SC cultures 30 min before NMDA exposure.
Inhibition of p38 MAPK but not p44/42 MAPK significantly reduced the
NMDA-induced LDH (Fig. 7A). In
addition, in pure microglial cultures, 10 mM SB203580 prevented
NMDA-induced microglial proliferation by 95% (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and NO metabolites 24 h after NMDA exposure. Our preliminary
results have shown that kainate and glutamate, but not agonists of
metabotropic glutamate receptors, are also able to trigger microglial
activation and proliferation (T. M. Tikka, B. L. Fiebich, and J.
E. Koistinaho, unpublished observations), suggesting that
agonists of ionotropic glutamate receptors are potential activators of
microglia. Several previous studies have suggested the involvement of
microglial activation in excitotoxicity (15, 18, 28),
although the mechanism is not yet fully understood. In the rat brain,
reactive microglia express GluR4 and NR1 subunits,
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and NMDAR subtypes,
respectively (29), and in vitro studies have demonstrated
the presence of group I metabotropic (30) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-kainate
glutamate receptors in pure microglia cultures (31). Noda
et al. (31) demonstrated that TNF- release from
cultured microglia is increased 2- to 3-fold 2 h after stimulation
with glutamate or kainate and by 45% after stimulation with
NMDA, even though only the responses to kainate and glutamate reached
statistical significance. Together with the previous studies, our
results support the idea that increased glutamate levels trigger
microglial proliferation and activation through ionotropic
receptors, including NMDAR, resulting in increased release of nitric
oxide and cytokines.
The second main finding was that microglia are able to enhance the
neurotoxicity of NMDA. We saw a robust increase in the number of
activated microglial cells as early as 1 h after administering
NMDA, whereas the LDH release, reflecting neuronal death, was
significantly elevated (only after 12–24 h. In addition, culturing
microglia on top of SC cultures enhanced the NMDA-induced LDH release.
Several in vivo studies have reported microglial proliferation and
activation early after various brain insults, including excitotoxin
applications (32). It is likely that the increased number
of OX-42-immunoreactive cells as soon as 1 h after NMDA
stimulation is due to activation and increased density of CD11b surface
Ag, whereas at later time points (12 and 24 h) increased
proliferation rate may well contribute to the severalfold increase
detected in the number of OX-42-immunoreactive cells. We hypothesize
that activated microglia enhance NMDA neurotoxicity through increased
release of IL-1 and NO, which is supported by the previous in vivo
and in vitro studies. First, application of kainate to the hippocampus
induces a rapid synthesis in IL-1 in microglia, resulting in
prolonged seizures and facilitation of glutamatergic function of the
NMDARs (32), possibly by attenuating astrocytic glutamate
uptake (33) and directly enhancing NMDAR function
(34). Second, blockade of IL-1 receptors or inhibition
of IL-1-converting enzyme provides neuroprotection against
excitotoxicity and ischemia (35, 36). Third, in
mixed neuronal cultures, combination of cytokines (IL-1 +
TNF-) induces neurotoxicity, which is reduced by blocking NO
production and NMDA antagonist (20). Finally, NMDA
neurotoxicity is mediated through NO, and inhibitors of NOS are
neuroprotective against ischemic insults (37, 38),
suggesting that the additional NO released by activated microglia may
well enhance NMDA-induced neuronal death. Our results stress the
importance of the role of the microglia in neuronal degeneration and
excitotoxicity, which is in accordance with the in vivo studies
demonstrating that the microglial activation is necessary but not
sufficient to trigger neuronal degeneration in the hippocampus after
excitotoxic insult (39). Our findings do not exclude the
possibility that release of other cytokines is involved in
microglia-enhanced excitotoxicity or that glutamate and still
uncharacterized factor(s) released from activated microglia enhance
excitotoxicity by acting through NMDARs (8, 28, 40).
The third major finding of our study is the neuroprotective role of minocycline, which may be based, at least partially, on the inhibition of microglia. This microglial inhibition included prevention of microglial proliferation in mixed SC and in pure microglia cultures and reduction of NO metabolite release and cytokine production in pure microglia cultures. Moreover, we found that minocycline was able to inhibit p38 MAPK in microglia and that specific inhibition of p38 MAPK, but not p44/42 MAPK, provided neuroprotection against NMDA toxicity and prevented microglial proliferation. p38 MAPK is thought to mediate inflammatory responses in various cell types (41, 42), including microglia (17); therefore, inhibition of p38 MAPK may be beneficial in injuries involving inflammation and microglial activation. Several studies with specific inhibitors of p38 MAPK have proved to be neuroprotective and provide antiinflammatory effects (17, 43, 44, 45). Another MAPK family member, p44/42, is stimulated by extracellular mitogens and is involved in proliferation and differentiation of several cell types, thereby supporting cellular survival (46). In agreement with the survival supporting role of p44/42 MAPK, Sugino et al. (44) did not observe neuroprotection against transient brain ischemia by inhibiting p44/42 MAPK, and in the present study inhibition of p44/42 MAPK slightly increased NMDA-induced neuronal death rather than being protective. However, p44/42 has also been shown to contribute to ischemic neuronal death in vivo (47) and in vitro (48), indicating that the role of p44/42 MAPK may depend on the injury model, timing, and dosing of the inhibitor treatment.
The specificity of SB203580, a p38 MAPK inhibitor, and PD98059, a
p44/42 MAPK inhibitor, has been widely studied and has been
demonstrated also in cultured microglia cells (19).
Importantly, Börsch-Haubold et al. (49) have shown
that at 20 µM concentrations these two inhibitors can directly
inhibit cyclooxygenase-1 and -2. Because substantially lower
concentrations were used in the present study and because in our cell
culture model there are no detectable levels of either isoform of
cyclooxygenase (27), it is very unlikely that the effects
of the inhibitors were even partially unspecific. Studies by SmithKline
Beecham Pharmaceuticals (Philadelphia, PA) have demonstrated that
SB203580 inhibits only p38 and p38 isoforms (50),
suggesting that p38
and p38
MAPK may not play a role in
microglial activation. It remains to be studied which p38 MAPK isoforms
are expressed in microglia.
Some studies have demonstrated that pretreatment with cytokines such as
TNF- and IL-1 provides neuroprotection against neuronal insults
(51, 52, 53, 54). However, pretreatment with sublethal injuries,
including brief ischemia or oxygen/glucose deprivation, is well known
to trigger tolerance against a subsequent neuronal insult, and this
preconditioning pathway involves NMDARs and nitric oxide
(55). It is therefore likely that, in addition to injury
model, the temporal relationship of increased cytokine or NO levels and
lethal neuronal insult determines, at least partially, the outcome
after excitotoxic injury.
Minocycline has been used in humans for several decades with tolerable side effects (56, 57). Considering the neuroprotection achieved with minocycline in animal models of stroke (24, 25) and Huntington’s disease (26), our cell culture findings suggest that minocycline may represent a potential therapeutic agent for treatment of brain diseases which involve excitotoxicity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jari Koistinaho, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. E-mail address: jari.koistinaho{at}uku.fi
3 Abbreviations used in this paper: NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; ICE, IL-1-converting enzyme; MAPK, mitogen-activated protein kinase; NOS, NO synthase; SC, spinal cord; HS-HI, horse serum heat-inactivated; LDH, lactate dehydrogenase.
Received for publication January 8, 2001. Accepted for publication April 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression in endotoxin-stimulated primary glial cultures. J. Neurosci. 18:1633. synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors. Brain Behav. Immun. 9:355.[Medline]
converting enzyme family protease reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA 94:2007. pretreatment induces protective effects against focal cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 17:483.[Medline]
, IL-1, IL-6, and TNF- impart neuroprotection to an excitotoxin through distinct pathways. J. Immunol. 163:3963. mRNA in ischemic brain tolerance. J. Neurosci. Res. 59:238.[Medline]
mRNA induction in ischemic brain tolerance by means of real-time polymerase chain reaction. J. Cereb. Blood Flow Metab. 20:15.[Medline]
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S. Franciosi, J. K. Ryu, H. B. Choi, L. Radov, S. U. Kim, and J. G. McLarnon Broad-Spectrum Effects of 4-Aminopyridine to Modulate Amyloid beta1-42-Induced Cell Signaling and Functional Responses in Human Microglia. J. Neurosci., November 8, 2006; 26(45): 11652 - 11664. [Abstract] [Full Text] [PDF]
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 C. C. Alano, T. M. Kauppinen, A. V. Valls, and R. A. Swanson Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations PNAS, June 20, 2006; 103(25): 9685 - 9690. [Abstract] [Full Text] [PDF]
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T. Malm, M. Ort, L. Tahtivaara, N. Jukarainen, G. Goldsteins, J. Puolivali, A. Nurmi, R. Pussinen, T. Ahtoniemi, T.-K. Miettinen, et al. beta-Amyloid infusion results in delayed and age-dependent learning deficits without role of inflammation or beta-amyloid deposits PNAS, June 6, 2006; 103(23): 8852 - 8857. [Abstract] [Full Text] [PDF]
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 H. Levkovitch-Verbin, M. Kalev-Landoy, Z. Habot-Wilner, and S. Melamed Minocycline delays death of retinal ganglion cells in experimental glaucoma and after optic nerve transection. Arch Ophthalmol, April 1, 2006; 124(4): 520 - 526. [Abstract] [Full Text] [PDF]
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M. A. Yenari, L. Xu, X. N. Tang, Y. Qiao, and R. G. Giffard Microglia Potentiate Damage to Blood-Brain Barrier Constituents: Improvement by Minocycline In Vivo and In Vitro Stroke, April 1, 2006; 37(4): 1087 - 1093. [Abstract] [Full Text] [PDF]
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 D. P. Stirling, K. M. Koochesfahani, J. D. Steeves, and W. Tetzlaff Minocycline as a Neuroprotective Agent Neuroscientist, August 1, 2005; 11(4): 308 - 322. [Abstract] [PDF]
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 F. Giuliani, W. Hader, and V. W. Yong Minocycline attenuates T cell and microglia activity to impair cytokine production in T cell-microglia interaction J. Leukoc. Biol., July 1, 2005; 78(1): 135 - 143. [Abstract] [Full Text] [PDF]
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 T. M. Kauppinen and R. A. Swanson Poly(ADP-Ribose) Polymerase-1 Promotes Microglial Activation, Proliferation, and Matrix Metalloproteinase-9-Mediated Neuron Death J. Immunol., February 15, 2005; 174(4): 2288 - 2296. [Abstract] [Full Text] [PDF]
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 D. C. Baptiste, A. T. E. Hartwick, C. A. B. Jollimore, W. H. Baldridge, G. M. Seigel, and M. E. M. Kelly An Investigation of the Neuroprotective Effects of Tetracycline Derivatives in Experimental Models of Retinal Cell Death Mol. Pharmacol., November 1, 2004; 66(5): 1113 - 1122. [Abstract] [Full Text] [PDF]
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 R. B. Rock, G. Gekker, S. Hu, W. S. Sheng, M. Cheeran, J. R. Lokensgard, and P. K. Peterson Role of Microglia in Central Nervous System Infections Clin. Microbiol. Rev., October 1, 2004; 17(4): 942 - 964. [Abstract] [Full Text] [PDF]
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 R.-R. Ji and G. Strichartz Cell Signaling and the Genesis of Neuropathic Pain Sci. Signal., September 28, 2004; 2004(252): re14 - re14. [Abstract] [Full Text] [PDF]
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J. Darman, S. Backovic, S. Dike, N. J. Maragakis, C. Krishnan, J. D. Rothstein, D. N. Irani, and D. A. Kerr Viral-Induced Spinal Motor Neuron Death Is Non-Cell-Autonomous and Involves Glutamate Excitotoxicity J. Neurosci., August 25, 2004; 24(34): 7566 - 7575. [Abstract] [Full Text] [PDF]
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 C. Zhang, B. Lei, T. T. Lam, F. Yang, D. Sinha, and M. O. M. Tso Neuroprotection of Photoreceptors by Minocycline in Light-Induced Retinal Degeneration Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2753 - 2759. [Abstract] [Full Text] [PDF]
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J. Wang, Q. Wei, C.-Y. Wang, W. D. Hill, D. C. Hess, and Z. Dong Minocycline Up-regulates Bcl-2 and Protects against Cell Death in Mitochondria J. Biol. Chem., May 7, 2004; 279(19): 19948 - 19954. [Abstract] [Full Text] [PDF]
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D. P. Stirling, K. Khodarahmi, J. Liu, L. T. McPhail, C. B. McBride, J. D. Steeves, M. S. Ramer, and W. Tetzlaff Minocycline Treatment Reduces Delayed Oligodendrocyte Death, Attenuates Axonal Dieback, and Improves Functional Outcome after Spinal Cord Injury J. Neurosci., March 3, 2004; 24(9): 2182 - 2190. [Abstract] [Full Text] [PDF]
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Y. D. Teng, H. Choi, R. C. Onario, S. Zhu, F. C. Desilets, S. Lan, E. J. Woodard, E. Y. Snyder, M. E. Eichler, and R. M. Friedlander Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury PNAS, March 2, 2004; 101(9): 3071 - 3076. [Abstract] [Full Text] [PDF]
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E. Diguet, C. E. Gross, E. Bezard, F. Tison, N. Stefanova, G. K. Wenning, B. Ravina, S. Fagan, R. Hart, C. Hovinga, et al. Neuroprotective agents for clinical trials in Parkinson's disease: A systematic assessment Neurology, January 13, 2004; 62(1): 158 - 159. [Full Text] [PDF]
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 R. M. Bonelli and H.-P. Kapfhammer Why Minocycline is Helpful in Huntington's Disease J Psychopharmacol, December 1, 2003; 17(4): 461 - 461. [PDF]
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 V. Raghavendra, F. Tanga, and J. A. DeLeo Inhibition of Microglial Activation Attenuates the Development but Not Existing Hypersensitivity in a Rat Model of Neuropathy J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 624 - 630. [Abstract] [Full Text] [PDF]
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 R. M. Friedlander Apoptosis and Caspases in Neurodegenerative Diseases N. Engl. J. Med., April 3, 2003; 348(14): 1365 - 1375. [Full Text] [PDF]
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R. M. Bonelli, C. Heuberger, and F. Reisecker Minocycline for Huntington's disease: An open label study Neurology, March 11, 2003; 60(5): 883 - 884. [Full Text] [PDF]
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T. M. Tikka, N. E. Vartiainen, G. Goldsteins, S. S. Oja, P. M. Andersen, S. L. Marklund, and J. Koistinaho Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease Brain, April 1, 2002; 125(4): 722 - 731. [Abstract] [Full Text] [PDF]
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D. C. Wu, V. Jackson-Lewis, M. Vila, K. Tieu, P. Teismann, C. Vadseth, D.-K. Choi, H. Ischiropoulos, and S. Przedborski Blockade of Microglial Activation Is Neuroprotective in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Mouse Model of Parkinson Disease J. Neurosci., March 1, 2002; 22(5): 1763 - 1771. [Abstract] [Full Text] [PDF]
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Y. Du, Z. Ma, S. Lin, R. C. Dodel, F. Gao, K. R. Bales, L. C. Triarhou, E. Chernet, K. W. Perry, D. L. G. Nelson, et al. Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease PNAS, November 20, 2001; (2001) 251341998. [Abstract] [Full Text] [PDF]
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Y. Du, Z. Ma, S. Lin, R. C. Dodel, F. Gao, K. R. Bales, L. C. Triarhou, E. Chernet, K. W. Perry, D. L. G. Nelson, et al. Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease PNAS, December 4, 2001; 98(25): 14669 - 14674. [Abstract] [Full Text] [PDF]
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