Hydroxyobtustyrene protects neuronal cells from chemical hypoxia-induced cell death

Article abstract of DOI:10.1007/s11418-018-1224-8

Hydroxyobtustyrene is a derivative of cinnamyl phenol isolated from Dalbergia odorifera T. Chen. The heartwood, known as ‘JiangXiang’, is a traditional Chinese medicine. Previous studies showed that hydroxyobtustyrene inhibited the biosynthesis of prostaglandins, which are mediators of neuronal cell death in ischemia. However, it currently remains unclear whether hydroxyobtustyrene protects neurons against ischemic stress. In the present study, we investigated the protective effects of hydroxyobtustyrene against sodium cyanide (NaCN)-induced chemical ischemia. Hippocampal neurons were cultured from the cerebral cortices of E18 Wistar rats. The effects of hydroxyobtustyrene on neuronal survival and trophic effects were estimated under lower and higher cell density conditions. After the treatment of 1?mM NaCN with or without hydroxyobtustyrene, an MTT assay, Hoechst staining, and immunocytochemistry for cyclooxygenase (COX)-2 were performed. Hydroxyobtustyrene increased cell viability under lower, but not normal density conditions. Neither the neurite number nor the length was influenced by hydroxyobtustyrene. NaCN significantly decreased viability and increased fragmentation in cell nuclei, and these changes were prevented by hydroxyobtustyrene. Moreover, NaCN increased the number of COX-2-positive neurons, and this was significantly prevented by the co-treatment with hydroxyobtustyrene. Therefore, hydroxyobtustyrene protected cultured hippocampal neurons against NaCN-induced chemical ischemia, which may be mediated by the inhibition of COX-2 production.

Full text of DOI:10.1007/s11418-018-1224-8

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1224-8
ORIGINAL PAPER
Hydroxyobtustyrene protects neuronal cells from chemical
hypoxia‑induced cell death
Takashi Iwai1,2,3 · Kengo Obara1 · Chihiro Ito4 · Hiroshi Furukawa4 · Jun‑Ichiro Oka1
Received: 27 January 2018 / Accepted: 29 June 2018
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Hydroxyobtustyrene is a derivative of cinnamyl phenol isolated from Dalbergia odorifera T. Chen. The heartwood, known as
‘JiangXiang’, is a traditional Chinese medicine. Previous studies showed that hydroxyobtustyrene inhibited the biosynthesis
of prostaglandins, which are mediators of neuronal cell death in ischemia. However, it currently remains unclear whether
hydroxyobtustyrene protects neurons against ischemic stress. In the present study, we investigated the protective efects of
hydroxyobtustyrene against sodium cyanide (NaCN)-induced chemical ischemia. Hippocampal neurons were cultured from
the cerebral cortices of E18 Wistar rats. The efects of hydroxyobtustyrene on neuronal survival and trophic efects were
estimated under lower and higher cell density conditions. After the treatment of 1 mM NaCN with or without hydroxy-
obtustyrene, an MTT assay, Hoechst staining, and immunocytochemistry for cyclooxygenase (COX)-2 were performed.
Hydroxyobtustyrene increased cell viability under lower, but not normal density conditions. Neither the neurite number nor
the length was infuenced by hydroxyobtustyrene. NaCN signifcantly decreased viability and increased fragmentation in cell
nuclei, and these changes were prevented by hydroxyobtustyrene. Moreover, NaCN increased the number of COX-2-positive
neurons, and this was signifcantly prevented by the co-treatment with hydroxyobtustyrene. Therefore, hydroxyobtustyrene
protected cultured hippocampal neurons against NaCN-induced chemical ischemia, which may be mediated by the inhibi-
tion of COX-2 production.
Keywords Ischemia · Cyclooxygenase · Dalbergia odorifera · Hippocampus · Apoptosis
Introduction
cerebral ischemia remains highly unsatisfactory. A number
of clinical trials on pharmacological neuroprotective strate-
gies for cerebral ischemia have resulted in disappointment
[2]. Therefore, the discovery of novel neuroprotective agents
for the treatment of cerebral stroke is urgently needed.
Cyanide is a well-known neurotoxin and select neurons
are particularly susceptible to its toxic efects [3]. A number
of patients who have survived acute cyanide intoxication
exhibit selective damage in the cortex, cerebellum, and basal
ganglia [4–7]. Cyanide inhibits the respiratory chain, the
utilization of oxygen, and mitochondrial functions [8, 9].
Since neurons are vulnerable to the loss of oxygen, cyanide
produces neuronal death by apoptosis or necrosis [10, 11].
Cyanide treatment (chemical ischemia) is commonly used as
an experimental model to study ischemia in vitro.
Cerebral ischemia is a devastating event that has become the
third largest cause of mortality and the leading cause of dis-
ability worldwide [1], and results in serious impairments in
central and peripheral functions. However, the treatment of
* Jun-Ichiro Oka
okaji@rs.noda.tus.ac.jp
1
Laboratory of Pharmacology, Faculty of Pharmaceutical
Sciences, Tokyo University of Science, 2641 Yamazaki,
Noda, Chiba 278-8510, Japan
2
Laboratory of Pharmacology, School of Pharmacy, Kitasato
University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641,
Japan
Cyclooxygenase (COX) is the rate-limiting enzyme in
prostaglandin (PG) production from arachidonic acid [12].
There are two isoforms of COX (COX-1 and COX-2) in the
brain [13]. COX-2 is expressed under ischemic conditions,
and enhances the production of PGs and reactive oxygen
3
Medicinal Research Laboratories, School of Pharmacy,
Kitasato University, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
4
Department of Medicinal Chemistry, Faculty of Pharmacy,
Meijo University, Tempaku-ku, Nagoya 468-8503, Japan
Vol.:(0123456789)
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Journal of Natural Medicines
species (ROS) as a side product [12]. Some of these prod-
ucts are toxic for neurons, and the inhibition of COX or
its downstream signals has been reported to protect neu-
rons in ischemia models including cyanide-induced chemi-
cal ischemia [14–18]. Therefore, COX and its downstream
molecules are potential targets in the treatment of ischemia.
Dalbergia odorifera T. Chen is a medium-sized evergreen
tree belonging to the Leguminosae family. Its heartwood,
known as ‘JiangXiang’, is a traditional Chinese medicine
and is widely used to dissipate blood stasis, stop bleeding,
and relieve pain, ischemia, swelling, necrosis, and rheu-
matic pain [19]. Previous studies reported that hydroxyob-
tustyrene, a derivative of cinnamyl phenol extracted from
D. odorifera, inhibited PG biosynthesis [20], and exerted
antioxidant efects (unpublished data). However, it currently
remains unknown whether hydroxyobtustyrene protects neu-
rons against ischemic stress. In the present study, we inves-
tigated the protective efects of hydroxyobtustyrene against
NaCN-induced chemical ischemia.
cells were treated for 24 h with 10 µM cytosine arabinoside
(Sigma) to remove actively dividing cells. The medium was
changed the next day (day 3), and subsequently twice per
week.
Synthesis of hydroxyobtustyrene
Hydroxyobtustyrene (Fig. 1) was synthesized as previously
reported [20]. 3-Methoxycatechol (700 mg) was reacted
with 3-phenyl-2-propen-1-ol (670 mg) in 90% formic acid
at 60 °C for 30 min to aford hydroxyobtustyrene (120 mg).
The yield of the product was approximately 9.4%. Hydroxy-
obtustyrene was dissolved in dimethyl sulfoxide (DMSO)
and stored at 4 °C. In our preliminary experiments using
cultured septal neurons, hydroxyobtustyrene increased cell
viability at a concentration of 100 or 300 nM, but not 10 nM.
Therefore, we used 100 nM hydroxyobtustyrene in the pre-
sent study.
Analysis of neurite length
Materials and methods
Immunocytochemistry for MAP-2 was performed with the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA,
USA). On day 3 and day 6, hydroxyobtustyrene (100 nM)
was added to the culture medium. Cells were fxed with
4% paraformaldehyde in 0.01 M phosphate-bufered saline
(PBS) (pH 7.5 at 4 °C) for 20 min. Fixed cells were blocked
with horse normal serum and incubated at 37 °C for 2 h with
a primary antibody (mouse anti-MAP-2 antibody, Sigma),
followed by an incubation with the secondary antibody at
37 °C for 30 min. Signals were detected with the ABC kit
and DAB kit (Vector Laboratories). Stained neurons were
imaged with a microscope (IX71; Olympus, Tokyo, Japan),
and the longest neurite length in each neuron was meas-
ured using the National Institutes of Health (NIH) ImageJ
software.
Ethics
All experimental protocols were approved by the Institu-
tional Animal Care and Use Committee at Tokyo University
of Science, and were conducted according to the guidelines
of the National Institute of Health and the Japan Neurosci-
ence Society.
Cell culture
Hippocampal neurons were cultured from the cerebral
cortices of E18 Wistar rats (Japan SLC Inc., Shizuoka,
Japan) under aseptic conditions as previously described
[21]. Briefy, brain tissue was cut into small pieces and
transferred to a centrifuge tube containing 0.25% trypsin
(Sigma) and 0.1% DNase 1 (Boehringer Mannheim, Man-
nheim, Germany) dissolved in DMEM (Gibco/Invitrogen,
Carlsbad, CA, USA). Tissue pieces were incubated at 37 °C
for 15 min with gentle agitation every 5 min and then gently
triturated using a fame-narrowed Pasteur pipette. The brain
tissue suspension was centrifuged at 1000 rpm for 5 min,
and the resultant pellet was resuspended in feeding medium,
which contained DMEM with 5% fetal bovine serum, 5%
horse serum, and 1% penicillin–streptomycin solution. The
cell suspension was passed through sterilized lens paper
before being diluted with feeding medium, and was then
seeded at 1.25×105 or 2.5×105 cells/cm2 on 24-well plastic
plates precoated with polyethylenimine (Sigma). Cells were
incubated at 37 °C in a 90 and 10% mixture of atmospheric
air and CO2, respectively. Two days after plating (day 2),
3‑[4,5‑Dimethlthiazol‑2‑yl]‑2,5‑diphenyltetrazolium
bromide (MTT) cell viability assay
On day 3 and day 6, NaCN (1 mM) with or without hydroxy-
obtustyrene (100 nM) was added to the culture medium for
24 h. After the 24-h treatment, neurons were washed with
PBS and then continuously cultured in normal medium. On
OH
HO
O
hydroxyobtustyrene
Fig. 1 Chemical structure of hydroxyobtustyrene
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Journal of Natural Medicines
day 9, MTT (0.5% fnal concentration, Wako) was added to
cultured neurons at 37 °C for 4 h. Formazan crystals were
dissolved in acidic isopropanol (0.04 M HCl in isopropanol),
and their absorbance was measured at 550 nm.
not under normal density conditions (2.5× 105 cells/cm2,
Fig. 2b). Under lower density conditions, the number of
neurites from soma (Fig. 2d) and the mean longest neurite
length (Fig. 2e) were not infuenced by the hydroxyobtus-
tyrene treatment.
DNA staining
Protective efects of hydroxyobtustyrene
on cultured hippocampal neurons against cell death
induced by chemical ischemia
NaCN (1 mM) with or without hydroxyobtustyrene (100 nM)
was added to the culture medium on day 4. After 24 h, DNA
staining was performed with Hoechst 33342 (Wako) in hip-
pocampal neurons [22]. The neurons were fxed with 4%
paraformaldehyde for 20 min. After two washes with PBS,
the neurons were incubated with Hoechst 33342 (5 µg/ml,
Wako) for 15 min at room temperature, and then observed
with a fuorescence microscope (IX71; Olympus).
In order to evaluate the efects of hydroxyobtustyrene on cell
viability in the case of chemical ischemia, MTT assays were
performed in 1 mM NaCN-treated neurons under normal cell
density conditions (Fig. 3). NaCN signifcantly decreased
cell viability, which was prevented by hydroxyobtustyrene
(Fig. 3).
COX‑2 immunostaining
In order to morphologically evaluate the protective efects
of hydroxyobtustyrene, DNA was visualized using Hoechst
staining (Fig. 4). After 24-h exposure to NaCN, the marked
fragmentation of cell nuclei was observed. The co-treatment
with hydroxyobtustyrene signifcantly decreased the frag-
mentation of nuclei. Hydroxyobtustyrene alone did not infu-
ence the morphology of nuclei or cell viability in the MTT
assay.
After the 4-h treatment with NaCN with or without hydroxy-
obtustyrene on day 4, the cells were fxed with 4% paraform-
aldehyde in 0.01 M PBS (pH 7.5) at 4 °C for 20 min. The
fxed cells were blocked with blocking solution (5% non-fat
milk, 1% bovine serum albumin in PBS), and were incubated
at 37 °C for 2 h with a primary antibody (goat anti-COX-2
antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA),
followed by an incubation with a secondary antibody (Alexa
488-conjugated anti-goat IgG, molecular probes) at 37 °C
for 30 min. Stained neurons were imaged with a microscope
(IX71; Olympus), and the stained area was measured using
NIH ImageJ software.
Hydroxyobtustyrene prevented COX‑2 expression
induced by chemical ischemia
We investigated whether hydroxyobtustyrene decreased
NaCN-induced COX-2 expression (Fig. 5). After 4-h expo-
sure to NaCN, the size of the COX-2-positive area mark-
edly increased. The co-treatment with hydroxyobtustyrene
signifcantly prevented the increase in the size of the COX-
2-positive area. Hydroxyobtustyrene alone did not infuence
the size of the COX-2-positive area.
Data analysis
Data are expressed as the mean SEM. The signifcance
of diferences was evaluated using a parametric one-way
analysis of variance followed by Bonferroni’s multiple com-
parison test. All statistical analyses were performed using
Graphpad Prism (Graphpad Software Inc., San Diego, CA,
USA). The criterion for signifcance was p<0.05 in all sta-
tistical evaluations.
Discussion
In the present study, we showed that hydroxyobtustyrene
prevented lower density- and chemical ischemia-induced
cell death in cultured hippocampal neurons. In addition,
hydroxyobtustyrene prevented COX-2 expression induced
by NaCN. Therefore, hydroxyobtustyrene may be a neuro-
protective substance against ischemic damage, and its efects
may be mediated by the inhibition of COX-2 expression.
The lower the cell density, the more difcult it is to
maintain cultures of primary neurons [23]. Neuronal death
under low density conditions is caused by a lack of parac-
rine trophic support from adjacent neurons and glia [24,
25]. Moreover, the lower the cell density, the greater the
reduction in the intracellular ATP concentration needed
to induce cell death following exposure to cellular stress
Results
Protective efects of hydroxyobtustyrene
on cultured hippocampal neurons against cell death
induced under low‑density conditions
In order to evaluate the efects of hydroxyobtustyrene on
cell viability and neurite length, MTT assays and MAP-2
staining were performed on hippocampal cultured neu-
rons. Hydroxyobtustyrene increased cell viability under
lower density conditions (1.25×105 cells/cm2, Fig. 2a), but
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Journal of Natural Medicines
1.25× 105 cells/cm2
2.5× 105 cells/cm2
a
b
Naive
Vehicle
Hydroxyobtustyrene
Naive
Vehicle
Hydroxyobtustyrene
2.5
2.0
1.5
1.0
0.5
0
**
1.5
1.0
0.5
0
ns
ns
ns
Hydroxyobtustyrene
Naive
Vehicle
c
d
Naive
Vehicle
Hydroxyobtustyrene
e
Naive
Vehicle
Hydroxyobtustyrene
ns
ns
3
2
1
0
2.0
1.5
1.0
0.5
0
ns
ns
Fig. 2 Hydroxyobtustyrene increased neural cell viability in a cell
density-dependent manner. Primary cultured rat neurons were
exposed to each compound containing medium for 6 days. a, b Cell
density-dependent neuronal viability assessed by the MTT assay
(n=4). c Representative images of MAP-2-like immunoreactivities
in primary cultured neurons (1.25×105 cells/cm2). d, e Quantifcation
of the number of neurites and neurite length per cell. Data are derived
from three independent experiments. Neurites were detected by
MAP2 immunostaining (n=4). Data were expressed as mean SEM.
**p<0.01, ns non-signifcant vs the vehicle group, one-way ANOVA
followed by Bonferroni’s test
[26]. Since hydroxyobtustyrene infuenced cell viability
under lower cell density conditions only, hydroxyobtusty-
rene may protect neurons from the lack of trophic support
or intracellular ATP. However, hydroxyobtustyrene itself
may have no neurotrophic efects, similar to brain-derived
neurotrophic factor, because neurite length was not infu-
enced by its treatment.
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Journal of Natural Medicines
1.5
Since ischemia is also reported to decrease the intracellu-
lar ATP, which leads the sequence of the pathophysiological
events [33], we hypothesized that hydroxyobtustyrene may
protect neurons from ischemic damage. We next investigated
the efects of hydroxyobtustyrene on chemical ischemia
induced by NaCN, and found neuroprotective efects in
the MTT assay. The active mitochondrial dehydrogenases
of living cells may cleave MTT to produce formazan, the
amount of which directly correlates with the number of
metabolically active cells [27]. However, the results of the
MTT assay are infuenced by endocytosis and the endocytic
processing of its reagents. Therefore, we morphologically
evaluated the protective effects of hydroxyobtustyrene
by Hoechst staining, and found that hydroxyobtustyrene
decreased the DNA fragmentation of neurons. These protec-
tive efects correlated with changes in the decrease in COX-2
positive neurons. In the brain, cyanide markedly increased
COX-2 expression and PG production in neurons, glia and
endothelial cells. In addition, the inhibition of COX or its
downstream signals (e.g., prostaglandin E2 EP3 receptor)
has been reported to protect neurons in ischemia models
[14, 16–18, 28]. Although the detailed mechanisms have
not yet been identifed, EP3 receptor activation is thought
to enhance neuronal cell death via the Rho kinase pathway
[18]. Our results that hydroxyobtustyrene decreased COX-2
expression in the present study agree with a previous report
demonstrating that hydroxyobtustyrene decreased PG pro-
duction [20]. Therefore, hydroxyobtustyrene may inhibit
downstream signals of COX-2 by inhibiting its expression,
which may, in turn, protect neurons against ischemic dam-
age. JiangXiang is used to treat pain and ischemia [19], in
which hydroxyobtustyrene may partially play a role. It is
necessary to demonstrate how hydroxyobtustyrene inhibits
chemical ischemia-induced COX-2 expression.
***
*
1.0
0.5
0.0
NaCN (1 mM)
Vehicle
Hydroxyobtustyrene (100 nM)
Fig. 3 Hydroxyobtustyrene rescued neuronal cell death induced by
chemical ischemia. Primary cultured rat neurons were exposed to
1 mM NaCN-containing medium for 6 days with or without 100 nM
hydroxyobtustyrene. Neuronal viability was assessed by the MTT
assay (n=30). Data were expressed as mean SEM. ***p<0.001,
*p<0.05 vs NaCN+the vehicle group, one-way ANOVA followed
by Bonferroni’s test
60
**
****
50
40
30
20
10
0
Prolonged dosing with a selective COX-2 inhibitor is
associated with an increased risk of cardiovascular events
(i.e., myocardial infraction and stroke) [29, 30]. This
increase in the risk of cardiovascular events is assumed to
be induced by a prostacyclin I2-thromboxane A2 imbalance
that leads to a prothrombotic state [31, 32]. Further studies
are needed in order to investigate the infuence of hydroxy-
obtustyrene on COX-1 expression and the product balance
of these mediators.
NaCN (1 mM)
Hydroxyobtustyrene (100 nM)
Vehicle
In conclusion, we demonstrated that hydroxyobtustyrene
exerted neuroprotective efects under lower cell density
and ischemic conditions. Its derivatives may be new candi-
dates for the treatment of ischemia and other infammatory
diseases.
Fig. 4 Hydroxyobtustyrene prevented NaCN-induced apopto-
sis in primary cultured neurons. Primary cultured rat neurons
(2.5×105 cells/cm2) were exposed to 1 mM NaCN-containing
medium for 24 h with or without 100 nM hydroxyobtustyrene.
Apoptotic cells were detected by Hoechst 33342 staining. Data were
expressed as mean SEM. (n=5) ****p<0.0001, **p<0.01 vs the
vehicle group, one-way ANOVA followed by Bonferroni’s test. Inset:
a representative image of an apoptotic cell indicated by the yellow
arrow. The apoptotic cell shows a shrunken and condensed morphol-
ogy
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Journal of Natural Medicines
Fig. 5 Hydroxyobtustyrene
decreased NaCN-induced COX-
2-positive cells in primary cul-
tured neurons. Primary cultured
rat neurons (2.5×105 cells/cm2)
were exposed to 1 mM NaCN-
containing medium for 4 h with
or without 100 nM hydroxyob-
tustyrene. a–d Representative
images of COX-2-positive cells.
e Quantifcation of the number
of COX-2-positive cells. Data
were expressed as mean SEM.
(n=5) ****p<0.0001 vs
a
b
Vehicle
NaCN + Vehicle
NaCN+the vehicle group,
c
d
one-way ANOVA followed by
Bonferroni’s test
NaCN + hydroxyobtustyrene
Hydroxyobtustyrene
e
25
20
15
10
5
****
****
0
NaCN (1 mM)
Vehicle
Hydroxyobtustyrene (100 nM)
Johns NE, Keiser J, King CH, Lozano R, Murdoch ME, O’Hanlon
S, Pion SD, Pullan RL, Ramaiah KD, Roberts T, Shepard DS,
Smith JL, Stolk WA, Undurraga EA, Utzinger J, Wang M, Mur-
ray CJ, Naghavi M (2014) The global burden of disease study
2010: interpretation and implications for the neglected tropical
diseases. PLoS Negl Trop Dis 8:e2865
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
2. Minnerup J, Sutherland BA, Buchan AM, Kleinschnitz C (2012)
Neuroprotection for stroke: current status and future perspec-
tives. Int J Mol Sci 13:11753–11772
References
3. Way JL (1984) Cyanide intoxication and its mechanism of
antagonism. Annu Rev Pharmacol Toxicol 24:451–481
1. Hotez PJ, Alvarado M, Basanez MG, Bolliger I, Bourne R, Bouss-
inesq M, Brooker SJ, Brown AS, Buckle G, Budke CM, Cara-
bin H, Cofeng LE, Fevre EM, Furst T, Halasa YA, Jasrasaria R,
1 3

Journal of Natural Medicines
4. Carella F, Grassi MP, Savoiardo M, Contri P, Rapuzzi B, Man-
goni A (1988) Dystonic-Parkinsonian syndrome after cyanide
poisoning: clinical and MRI fndings. J Neurol Neurosurg Psy-
chiatry 51:1345–1348
20. Goda Y, Kiuchi F, Shibuya M, Sankawa U (1992) Inhibitors of
prostaglandin biosynthesis from Dalbergia odorifera. Chem
Pharm Bull (Tokyo) 40:2452–2457
21. Iwai T, Ito S, Tanimitsu K, Udagawa S, Oka J (2006) Glucagon-
like peptide-1 inhibits LPS-induced IL-1β production in cultured
rat astrocytes. Neurosci Res 55:352–360
5. Valenzuela R, Court J, Godoy J (1992) Delayed cyanide induced
dystonia. J Neurol Neurosurg Psychiatry 55:198–199
6. Rosenow F, Herholz K, Lanfermann H, Weuthen G, Ebner R,
Kessler J, Ghaemi M, Heiss WD (1995) Neurological sequelae of
cyanide intoxication—the patterns of clinical, magnetic resonance
imaging, and positron emission tomography fndings. Ann Neurol
38:825–828
22. Iwai T, Iinuma Y, Kodani R, Oka J (2008) Neuromedin U inhibits
infammation-mediated memory impairment and neuronal cell-
death in rodents. Neurosci Res 61:113–119
23. Yu X, An L (2002) A serum- and antioxidant-free primary cul-
ture model of mouse cortical neurons for pharmacological screen
and studies of neurotrophic and neuroprotective agents. Cell Mol
Neurobiol 22:197–206
7. Rosenberg NL, Myers JA, Martin WR (1989) Cyanide-induced
parkinsonism: clinical, MRI, and 6-fuorodopa PET studies. Neu-
rology 39:142–144
24. Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat
Protoc 1:2406–2415
8. Gunasekar PG, Sun PW, Kanthasamy AG, Borowitz JL, Isom GE
(1996) Cyanide-induced neurotoxicity involves nitric oxide and
reactive oxygen species generation after N-methyl-d-aspartate
receptor activation. J Pharmacol Exp Ther 277:150–155
9. Bhattacharya R, Lakshmana Rao PV (2001) Pharmacological
interventions of cyanide-induced cytotoxicity and DNA damage
in isolated rat thymocytes and their protective efcacy in vivo.
Toxicol Lett 119:59–70
25. Kaneko A, Sankai Y (2014) Long-term culture of rat hippocampal
neurons at low density in serum-free medium: combination of the
sandwich culture technique with the three-dimensional nanof-
brous hydrogel PuraMatrix. PLoS One 9:e102703
26. Fujita R, Yoshida A, Mizuno K, Ueda H (2001) Cell density-
dependent death mode switch of cultured cortical neurons under
serum-free starvation stress. Cell Mol Neurobiol 21:317–324
27. Zhou J, Tang XC (2002) Huperzine A attenuates apoptosis and
mitochondria-dependent caspase-3 in rat cortical neurons. FEBS
Lett 526:21–25
10. Mills EM, Gunasekar PG, Li L, Borowitz JL, Isom GE (1999) Dif-
ferential susceptibility of brain areas to cyanide involves diferent
modes of cell death. Toxicol Appl Pharmacol 156:6–16
11. Prabhakaran K, Li L, Borowitz JL, Isom GE (2002) Cyanide
induces diferent modes of death in cortical and mesencephalon
cells. J Pharmacol Exp Ther 303:510–519
28. Collino M, Aragno M, Mastrocola R, Gallicchio M, Rosa AC,
Dianzani C, Danni O, Thiemermann C, Fantozzi R (2006) Mod-
ulation of the oxidative stress and infammatory response by
PPAR-γ agonists in the hippocampus of rats exposed to cerebral
ischemia/reperfusion. Eur J Pharmacol 530:70–80
12. Vane JR, Bakhle YS, Botting RM (1998) Cyclooxygenases 1 and
2. Annu Rev Pharmacol Toxicol 38:97–120
29. Topol EJ (2004) Failing the public health—rofecoxib, Merck, and
the FDA. N Engl J Med 351:1707–1709
13. Smith WL, DeWitt DL, Garavito RM (2000) Cyclooxygenases:
structural, cellular, and molecular biology. Annu Rev Biochem
69:145–182
30. Solomon SD, McMurray JJ, Pfefer MA, Wittes J, Fowler R, Finn
P, Anderson WF, Zauber A, Hawk E, Bertagnolli M, Adenoma
Prevention with Celecoxib Study I (2005) Cardiovascular risk
associated with celecoxib in a clinical trial for colorectal adenoma
prevention. N Engl J Med 352:1071–1080
14. Gunasekar PG, Borowitz JL, Isom GE (1998) Cyanide-induced
generation of oxidative species: involvement of nitric oxide syn-
thase and cyclooxygenase-2. J Pharmacol Exp Ther 285:236–241
15. Ohanian SH, Borsos T (1975) Lysis of tumor cells by antibody
and complement. II. Lack of correlation between amount of C4
and C3 fxed and cell lysis. J Immunol 114:1292–1295
16. Kawano T, Anrather J, Zhou P, Park L, Wang G, Frys KA, Kunz
A, Cho S, Orio M, Iadecola C (2006) Prostaglandin E2 EP1 recep-
tors: downstream efectors of COX-2 neurotoxicity. Nat Med
12:225–229
31. FitzGerald GA (2003) COX-2 and beyond: approaches to pros-
taglandin inhibition in human disease. Nat Rev Drug Discov
2:879–890
32. Hewett SJ, Bell SC, Hewett JA (2006) Contributions of cycloox-
ygenase-2 to neuroplasticity and neuropathology of the central
nervous system. Pharmacol Ther 112:335–357
33. Rodrigo R, Fernández-Gajardo R, Gutiérrez R, Matamala JM,
Carrasco R, Miranda-Merchak A, Feuerhake W (2013) Oxidative
stress and pathophysiology of ischemic stroke: novel therapeutic
opportunities. CNS Neurol Disord Drug Targets 12:698–714
17. Candelario-Jalil E, Fiebich BL (2008) Cyclooxygenase inhibition
in ischemic brain injury. Curr Pharm Des 14:1401–1418
18. Ikeda-Matsuo Y, Tanji H, Narumiya S, Sasaki Y (2011) Inhibition
of prostaglandin E2 EP3 receptors improves stroke injury via anti-
infammatory and anti-apoptotic mechanisms. J Neuroimmunol
238:34–43
19. Sun S, Zeng X, Zhang D, Guo S (2015) Diverse fungi associated
with partial irregular heartwood of Dalbergia odorifera. Sci Rep
5:8464
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