Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia

Article abstract of DOI:10.1016/j.ejmech.2018.11.060

Ischemia-reperfusion injuries produce reactive oxygen species that promote the peroxide lipid oxidation process resulting in the production of an endogenic lipid peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces cell de

Full text of DOI:10.1016/j.ejmech.2018.11.060

Accepted Manuscript
Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal
CA1 damage after transient cerebral ischemia
Kei Noguchi, Taha F.S. Ali, Junko Miyoshi, Kimihiko Orito, Tetsuya Negoto, Tanima
Biswas, Naomi Taira, Ryoko Koga, Yoshinari Okamoto, Mikako Fujita, Masami
Otsuka, Motohiro Morioka
PII:
S0223-5234(18)31019-5
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.060
EJMECH 10918
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 August 2018
Accepted Date: 23 November 2018
Please cite this article as: K. Noguchi, T.F.S. Ali, J. Miyoshi, K. Orito, T. Negoto, T. Biswas, N. Taira,
R. Koga, Y. Okamoto, M. Fujita, M. Otsuka, M. Morioka, Neuroprotective effects of a novel carnosine-
hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia, European Journal
of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.060.
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Neuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1
damage after transient cerebral ischemia
Kei Noguchi, MD†1; Taha F.S. Ali†2,3; Junko Miyoshi, MD1; Kimihiko Orito, MD1;
Tetsuya Negoto, MD1;Tanima Biswas2; Naomi Taira2; Ryoko Koga2; Yoshinari Okamoto2;
Mikako Fujita4; Masami Otsuka2; Motohiro Morioka, MD*1
1Department of Neurosurgery, Kurume University, School of Medicine, Fukuoka, Japan
2Department of Bioorganic and Medicinal Chemistry, Kumamoto University,
Kumamoto, Japan
3Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt
4Research Institute for Drug Discovery, Kumamoto University, Kumamoto, Japan
†These authors contributed equally to this work
*Corresponding author
Address correspondence and reprint requests to:
Motohiro Morioka
Department of Neurosurgery
Kurume University School of Medicine
67, Asahimachi Kurume City, Fukuoka 830-0011, Japan
Telephone: +81-942-31-7570
Fax: +81-942-38-8179

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E-mail address: mmorioka@med.kurume-u.ac.jp
Cover title: Neuroprotective effects of L-carnosine hydrazide
Figure count: 6
Word count: 5,161
Search terms: Animal Models of Human Disease, Ischemic Stroke, Oxidant Stress
Abbreviations:
bilateral common carotid artery occlusion = BCCO; heat shock protein 70 = Hsp70; histidyl
hydrazide = HH; L-Carnosine = L-CAR; L-carnosine hydrazide = CNN; reactive carbonyl
species = RCS; 4-hydroxy-trans-2-nonenal = 4-HNE; reactive oxygen species = ROS;
phosphate-buffered saline = PBS; delayed neuronal death = DND; carnosine-NHNH-Ac =
CNNA; Ac-carnosine-NHNH2 = ACNN; Ac-carnosine-NHNH-Ac = ACNNA; high-power
field = HPF; attenuated total reflection = ATR; nuclear magnetic resonance = NMR

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Abstract
Ischemia-reperfusion injuries produce reactive oxygen species that promote the
peroxide lipid oxidation process resulting in the production of an endogenic lipid
peroxide, 4-hydroxy-trans-2-nonenal (4-HNE), a highly cytotoxic aldehyde that induces
cell death. We synthesized a novel 4-HNE scavenger – a carnosine-hydrazide derivative,
L-carnosine hydrazide (CNN) – and examined its neuroprotective effect in a model of
transient ischemia.
PC-12 cells were pre-incubated with various doses (0-50 mmol/L) of CNN for 30 min,
followed by incubation with 4-HNE (250 µM). An MTT assay was performed 24 h later
to examine cell survival. Transient ischemia was induced by bilateral common carotid
artery occlusion (BCCO) in the Mongolian gerbil. Animals were assigned to
sham-operated (n = 6), placebo-treated (n = 12), CNN pre-treated (20 mg/kg; n = 12),
CNN post-treated (100 mg/kg; n = 11), and histidyl hydrazide (a previously known
4-HNE scavenger) post-treated (100 mg/kg; n = 7) groups. Heat shock protein 70
immunoreactivity in the hippocampal CA1 region was evaluated 24 h later, while
delayed neuronal death using 4-HNE staining was evaluated 7 days later.
Pre-incubation with 30 mmol/L CNN completely inhibited 4-HNE-induced cell toxicity.
CNN prevented delayed neuronal death by >60% in the pre-treated group
(p < 0.001)
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and by >40% in the post-treated group (p < 0.01). Histidyl hydrazide post-treatment
elicited no protective effect. CNN pre-treatment resulted in high heat shock protein 70
and low 4-HNE immunoreactivity in CA1 pyramidal neurons. Higher 4-HNE
immunoreactivity was also found in the placebo-treated animals than in the CNN
pre-treated animals.
Our novel compound, CNN, elicited highly effective 4-HNE scavenging activity in vitro.
Furthermore, CNN administration both pre- and post-BCCO remarkably reduced
delayed neuronal death in the hippocampal CA1 region via its induction of heat shock
protein 70 and scavenging of 4-HNE.
Keywords: carnosine hydrazide, free radical scavenger, global cerebral ischemia,
Mongolian gerbils, hippocampus
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1. Introduction
The purpose of neuroprotective therapy during the acute stages of cerebral ischemia is
to block cytotoxic processes and protect neurons against toxic molecules to prevent cell
death. Reactive carbonyl species (RCS) are cytotoxic substances generated from
oxidative stress, including membrane lipid-peroxidation reactions, caused by
ischemia/reperfusion injuries in the brain [1]. RCS cause damage to proteins, nucleic
acids, and lipids, the chemical modification of which results in cytotoxicity and
mutagenicity. In addition to its direct toxic effects, the modification of biomolecules by
RCS gives rise to a multitude of adducts and cross-linked proteins that have
increasingly been implicated in aging and the pathology of a wide range of human
diseases [2,3]. RCS can be classified into three groups: (1) α, β-unsaturated aldehydes,
such as 4-hydroxy-trans-2-nonenal (4-HNE) and acrolein; (2) keto-aldehydes, such as
methylglyoxal; and (3) dialdehydes, such as glyoxal and malondialdehyde [4]. Among
these RCS, 4-HNE represents one of the most abundant and highly cytotoxic aldehydes
generated during lipid peroxidation. For example, it has been reported that 4-HNE
greatly contributes to the delayed neuronal death (DND) of pyramidal cells in the CA1
region of the hippocampus after transient cerebral ischemia [5-7]. Although the cell
death cascade might start earlier, morphological DND occurs 4-7 days after ischemic
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stroke in the hippocampal CA1 region and ultimately induces DNA fragmentation via
apoptotic processes [8-12]. Although this phenomenon is clearly different from necrosis
(real infarction; core of infarction) caused by severe ischemia, similar processes can be
visualized in brain areas surrounding the necrotic tissue. Thus, various types of
4-HNE-scavenging substances have been examined in attempts to prevent DND.
L-Carnosine (L-CAR, Fig. 1), a natural β-alanyl-L-histidine dipeptide that is widely
distributed in skeletal muscle and the central nervous system, was first isolated from
skeletal muscle extracts by Gulewitsch et al. at the end of the 19th century [13,14]. In
biological tissues, L-CAR acts as an antioxidant and scavenger of RCS; as such, L-CAR
may protect tissue from oxidative damage [15,16]. It has been proposed that since
L-CAR acts as a natural scavenger of cytotoxic aldehydes, it could be a potent and
selective scavenger of the toxic lipid peroxidation product, 4-HNE [17,19]. In contrast,
several papers have reported that the histidine analogue, L-histidyl hydrazide
(histidyl-NHNH2, HH, Fig. 1), is more efficient than L-CAR in 4-HNE scavenging and
in reducing brain damage [20,21]. The formation of cyclic adduct, L-CAR-4-HNE and
HH-4-HNE, is presumed (Fig.1). In this study, we integrated the functional groups of
L-CAR and HH (i.e., amino and imidazole groups and hydrazide and imidazole groups,
respectively) into one molecule to form L-carnosine hydrazide (L-carnosine-NHNH2,
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CNN), expecting the formation of a tighter bicyclic adduct (Fig. 1). We also designed
acetyl derivatives ACNN, CNNA, and ACNNA to investigate the role of the amino and
the hydrazide groups of CNN (Fig. 1).
Fig. 1: Structures of L-carnosine (L-Car), L-histidine (L-His), histidyl-NHNH2 (HH),
L-carnosine hydrazide (CNN), and the acetylamino derivatives, Ac-carnosine-NHNH2
(ACNN); acetylhydrazide derivative, carnosine-NHNH-Ac (CNNA);
acetylamino-acetylhydrazide derivative, Ac-carnosine-NHNH-Ac (ACNNA);
L-carnosine (L-CAR) together with the proposed structure of the 4-HNE adducts.
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2. Results and discussion
2.1. Chemistry
Compounds were prepared as shown in Scheme 1 and 2. L-carnosine was esterified,
acetylated and treated with hydrazine hydrate to give ACNN (Scheme 1). DCC
condensation of Cbz-β-Ala and His-OMe afforded Cbz-carnosine-Ome which was
treated with hydrazine hydrate to give Cbz-carnosine-NHNH2 (Scheme 2). Removal of
Cbz group of Cbz-carnosine-NHNH2 gave CNN that was further acetylated to give
diacetylated ACNNA. Acetylation of Cbz-carnosine-NHNH2 followed by
hydrogenolysis gave monoacetylated CNNA. Detail of the synthesis is given in
Supplementary Material.
Scheme 1. Synthesis of ACNN.
a) SOCl2, MeOH, 0 °C 10 min then reflux 1 h, b) Ac2O, MeOH, KHCO3, 0 °C 30 min
then rt overnight, c) N2H4･H2O, EtOH, rt overnight.
Scheme 2. Syntheses of CNN, ACNNA, and CNNA.
a) DCC, CH2Cl2, 0 °C 2 h then rt overnight, b) N2H4･H2O, EtOH, rt overnight, c) H2,
10% Pd/C, MeOH, rt overnight, d) Ac2O, MeOH, DIPEA, 0 °C 30 min then rt
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overnight.
2.2. Measurement of 4-HNE-quenching activity
The proportion of 4-HNE-quenching activity was calculated by comparing the 4-HNE
consumption achieved by each compound with that achieved by CNN (defined as
100%; Fig. 2A, B). Fig. 2A presents the 4-HNE capturing activity of each compound
tested after 1 h of incubation. CNN, HH, and carnosine-NHNH-Ac (CNNA) were more
potent than L-CAR, while Ac-carnosine-NHNH2 (ACNN) was less active than L-CAR,
and Ac-carnosine-NHNH-Ac (ACNNA) elicited the lowest activity. These findings
suggest that the introduction of an acetyl group masked the activity of CNN,
demonstrating the significance of the amino and hydrazide groups. Moreover, the amino
group may be more important than the hydrazide group since ACNN was less active
than CNNA, but the masked hydrazide group elicited some activity because CNNA was
more active than L-CAR.
Fig. 2B depicts the time course of 4-HNE-quenching activity by CNN, L-CAR, and HH.
While CNN and HH demonstrated similar 4-HNE-capturing rates 1 h after incubation
(Fig. 2A), CNN was clearly superior since, after 30 min of incubation, the amount of
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4-HNE captured by CNN, HH, and L-CAR corresponded to approximately 90%, 70%,
and 20%, respectively.
Fig. 2: Depiction of the 4-hydroxy-trans-2-nonenal (4-HNE)-quenching activity of
L-carnosine hydrazide (CNN), Ac-carnosine-NHNH2 (ACNN), carnosine-NHNH-Ac
(CNNA), Ac-carnosine-NHNH-Ac (ACNNA), L-carnosine (L-CAR), L-histidine, and
histidyl hydrazide (HH). (A) The 4-HNE-capturing activity of all compounds after 1 h
incubation. (B) Time course measurement of the 4-HNE-quenching activity of CNN,
L-CAR, and HH.
2.3. Cell culture and MTT assay
Fig. 3 illustrates the protective effect of CNN against 4-HNE in PC-12 cells in vitro.
Pre-incubated (30 min) CNN did not elicit cell toxicity. Furthermore, CNN was able to
penetrate cells, and elicit protective effects in a dose-dependent manner. Additionally,
30 mM CNN elicited a complete protective effect against 250 µM 4-HNE.
Fig. 3: Effect of L-carnosine hydrazide on 4-hydroxy-trans-2-nonenal-induced PC-12
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cell death
Cells were treated with various concentrations of L-carnosine hydrazide (CNN) for 30
min followed by 1 day of 4-hydroxy-trans-2-nonenal (4-HNE) exposure. Results from
the MTT assays are presented (data from 3-6 different experiments) as means ± standard
deviations. # p < 0.05 compared to PC-12 cells exposed to 4-HNE without CNN (n = 4);
## p = 0.052 compared to PC-12 cells exposed to 4-HNE without CNN (n = 4).
2.4. Protective effect of CNN against DND
DND was clearly observed in the CA1 subfield of the hippocampus 7 days after
ischemia (Fig. 4 and 5). More specifically, the number of viable neurons was 70.3 ±
4.5 per high-power field (HPF) in the sham-operated group versus 1.4 ± 1.3 per HPF in
the placebo-treated group, revealing a statistically significant loss of neurons following
bilateral common carotid artery occlusion (BCCO) (p < 0.05). Significantly more viable
neurons were observed in the CNN pre-administration group (44.9 ± 17.7 per HPF) than
in the placebo-treated group (p < 0.05; Fig. 4). A neuroprotective effect was also
observed in the CNN post-administration group (24.9 ± 25.1 neurons/HPF, p < 0.05),
although this effect was weaker than that observed in the pre-administration group (Fig.
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4D and Fig. 5). In contrast, no significant difference was observed between the HH
post-administration and placebo-treated groups (7.4 ± 15.4 neurons/HPF; Fig. 4 and 5).
Together, these findings indicated that, while both CNN administration groups exhibited
statistically significant neuroprotective activity, CNN pre-administration tended to be
more effective than CNN post-administration. Specifically, pre-administration of CNN
elicited a >60% protective effect against DND versus the 40% observed in the
post-administration group.
Fig. 4: Neuroprotective effect of L-carnosine hydrazide on the pyramidal neuron layer
in the middle CA1 region of the hippocampus 7 days after bilateral common carotid
artery occlusion
Sections stained with hematoxylin-eosin from (A) sham-operated, (B) placebo-treated,
(C) L-carnosine hydrazide (CNN)-treated pre-bilateral common carotid artery occlusion
(BCCO), (D) CNN-treated post-BCCO, and (E) histidyl hydrazide (HH)-treated
post-BCCO animals. Magnification 400×, scale bar 20 µm.
Fig. 5: Histogram representing the number of viable CA1 pyramidal neurons
(neurons/high-power field) in each treatment group. Statistical differences were
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determined with a Student’s t-test. *** p < 0.001 compared to the sham-operated group;
### p < 0.001 compared to the placebo-treated group; ## p < 0.01 compared to the
placebo-treated group. Sham = sham-operated group; placebo = placebo-treated group;
CNN pre = L-carnosine hydrazide pre-administration group; CNN post = L-carnosine
hydrazide post-administration group; HH post = histidyl hydrazide post-administration
group. Each value is expressed as mean ± SD.
2.5. Evaluation of immunostaining
Representative photographs of 4-HNE and heat shock protein 70 (Hsp70)
immunostaining in the hippocampal CA1 region at 24 h after BCCO induction are
presented in Fig. 6. Higher 4-HNE immunoreactivity was observed in the cytoplasm of
pyramidal neurons in the placebo-treated group than in the CNN pre-administration
group. In contrast, the CNN pre-administration group exhibited higher concentrations of
Hsp70 immunoreactivity in the cell cytoplasm of pyramidal neurons than did the
placebo-treated group.
Fig. 6: Representative photograph of 4-hydroxy-trans-2-nonenal and heat shock protein
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70 immunostaining of the pyramidal neuron layer in the middle CA1 region of the
hippocampus 24 h after bilateral common carotid artery occlusion
Higher 4-hydroxy-trans-2-nonenal (4-HNE) immunoreactivity was observed in the
cytoplasm of pyramidal neurons in tissue from a placebo-treated animal than in that
from an animal that received L-carnosine hydrazide (CNN) prior to bilateral common
carotid artery occlusion (BCCO) (arrow head). CNN pre-administration resulted in
higher heat shock protein 70 (Hsp70) immunoreactivity than did placebo treatment.
Sections were stained with anti-4-HNE antibody and anti-Hsp70 antibody.
Magnification 400×, scale bar 20 µm.
2.6. Discussion
Our study demonstrated that the novel derivative, CNN, had significant neuroprotective
effects against DND (by scavenging 4-HNE and preserving Hsp70 induction) in the
hippocampal CA1 region of Mongolian gerbils following temporary bilateral occlusion
of the common carotid artery.
We synthesized three new derivatives of CNN, namely ACNN, CNNA, and ACNNA
(Fig. 1), and confirmed that CNN had the most potent 4-HNE-quenching activity. The
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superiority of CNN was due to the the reactivity of the amino and hydrazide groups to
form a covalent bond with 4-HNE. We found that CNN was more potent than the
previously well-known scavengers, HH, L-histidine, and L-CAR (Fig. 2) [17-23].
The efficacy of HH in protecting neurons against 4-HNE toxicity has been demonstrated
in several papers. For example, Guiotto et al. reported that the histidine analogue, HH,
is more efficient than carnosine in eliciting aldehyde-sequestering effects and in
protecting SH-SY5Y neuroblastoma cells and rat hippocampal neurons from
4-HNE-mediated death [21]. Tang et al. also reported HH as being effective in reducing
brain damage and improving functional outcome in a mouse model of focal ischemic
stroke [20]. In the current study, we found that the 4-HNE-quenching activities of CNN
and HH were similar after 60 min of incubation (Fig. 2A). However, after only 20 min
of incubation, the activity of CNN was remarkably higher than that of HH (e.g. 80% of
CNN vs 60% of HH, Fig. 2B). The reason for this disparity likely lies in the fact that
CNN is able to rapidly and tightly react with 4-HNE to form a bicyclic adduct using its
three functional groups (i.e., amino, imidazole, and hydrazide), while HH captures
4-HNE gradually and loosely with only “two hands” (imidazole and hydrazide). This
difference in reaction rate is important, because RCS are generated rapidly after
ischemic insult. Indeed, although HH exhibited high 4-HNE scavenging activity
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(similar to CNN after 60 min incubation) in vitro, no difference in scavenging activity
was observed when animals were post-treated with HH in our in vivo study. Together,
these findings suggest that pre-administration of HH may be effective against DND;
however, since we aimed to apply our novel CNN derivatives to actual clinical
conditions (i.e., after the onset of ischemia), we did not evaluate HH prior to the
induction of BCCO. In contrast, CNN elicited a significant neuroprotective effect
against DND, even after the onset of ischemia; this result is of great clinical
significance.
Alongside our evaluation of 4-HNE-quenching activity, we examined 4-HNE and
Hsp70 immunostaining to elucidate how CNN prevented DND. Previously, 4-HNE was
proposed to be an important marker of radical-induced lipid peroxidation during
cerebral post-ischemic reperfusion injury [17-19,24]. Hsp70 is expressed at low levels
in the normal brain but is upregulated under conditions of oxidative stress, such as in
the penumbra during the hyperacute stage of stroke [25,26]. Hsp70 is well known for its
cytoprotective role against acute ischemic stroke [27,28], and the possibility of a
correlation between Hsp70 expression and increased tolerance to ischemia has been
reported [29-32]. In the current study, we found that, compared to placebo treatment,
CNN administration reduced 4-HNE and increased Hsp70 immunoreactivity. This result
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might be explained by the well-established “calpain-cathepsin hypothesis” [33-36],
which states that ischemia induces Ca2+ mobilization and Ca2+-induced calpain
activation. Calpain plays a major role in lysosomal rupture, and lipid peroxidation after
ischemic re-perfusion produces 4-HNE that carbonylates Hsp70 at the lysosomal
membrane. Carbonylation of Hsp70 leads to not only loss of its chaperone function, but
also vulnerability to calpain cleavage. Lysosomal membrane damage is most remarkable
when Ca2+-induced calpain activation and 4-HNE-induced Hsp70 carbonylation
function in parallel. Consequently, the loss of normal (non-carbonylated) Hsp70 results
in the rupture or permeabilization of lysosomal membranes and cathepsin release. These
reactions all occur specifically in the CA1 region of the hippocampus and can be the
trigger of CA1-related neuronal death.
The limitation of CNN is the relatively high dose 100 to 200 mg/kg in animal
experiments. This could be overcome by structure modification of CNN to improve the
brain migration property.
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3. Conclusion
This study demonstrated that our newly developed carnosine-hydrazide derivative
elicited high 4-HNE-scavenging activity, with CNN exhibiting highly effective
scavenging activity in vitro. CNN administration in Mongolian gerbils also remarkably
reduced DND in the CA1 region of the hippocampus after transient cerebral ischemia
induced by temporary BCCO. The highly effective scavenging ability of CNN could be
due to its ability to prevent 4-HNE-induced Hsp70 carbonylation. As 4-HNE has been
implicated in many neurodegenerative disorders, these novel derivatives may be
effective in treating a number of diseases associated with 4-HNE pathogenesis, such as
Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and
cardiovascular diseases such as atherosclerosis and stroke [20,37-41]. We are currently
developing CNN derivatives to improve the pharmacokinetic property and to extend the
utility of CNN.
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4. Methods
4.1. General information
All procedures in this study were carried out in strict accordance with the Welfare and
Management of Animals Act (Act No. 105 of 1973); standards relating to the Care and
Keeping and Reducing Pain of Laboratory Animals (Notice of the Ministry of the
Environment No. 84 of 2013); Fundamental Guidelines for Proper Conduct of Animal
Experiments and Related Activities in Academic Research Institutions under the
jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology
(Notice of the Ministry of Education, Culture, Sports, Science and Technology, No. 71
of 2006); Guidelines for Proper Conduct of Animal Experiments (Science Council of
Japan, 2006); Methods of Destruction of Animals (Notice of the Ministry of the
Environment No. 105 of 2007); and the guidelines for animal experimentation from the
Animal Research Center of Kurume University.
All animal experiments were reviewed by the Institutional Animal Care and Use
Committee and approved by the president of Kurume University (IACUC #2016-172,
#2017-033). All induction was performed under anesthesia, and all efforts were made to
minimize suffering.
Statistical analysis was performed using JMP® Pro13 software (SAS Institute Inc., Cary,
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NC, USA). A Student’s t-test was applied to compare the number of surviving neurons
in the CA1 of each of the five groups. Data are presented as mean ± standard deviation
(SD). A p-value of < 0.05 was considered significant.
4.2. Animals
Adult male Mongolian gerbils (MON/JmsGbs, Nihon SLC Co., Shizuoka, Japan),
weighting 60-100 g, were used for all experiments. The gerbils were kept in a controlled
environment (22 ± 2°C, 55 ± 5% humidity) and both laboratory food and tap water
were available ad libitum. The colony was maintained on a 12 h light/dark cycle, with
lights on between 7:00 and 19:00.
4.3. Drugs
In addition to CNN, we designed three acetyl-masked derivatives to study the role of the
amino and hydrazide groups, i.e., the acetylamino derivative, ACNN, the
acetylhydrazide derivative, CNNA, and the acetylamino-acetylhydrazide derivative,
ACNNA (Fig. 1). These compounds were synthesized as described in Scheme 1, 2, and
the Supplementary Data Material. The structures of these derivatives were confirmed
and their purity was confirmed by high performance liquid chromatography (96-100%).
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4.4. Measurement of 4-HNE-quenching activity
In this study, 4-HNE-quenching activity was evaluated by measuring 4-HNE
consumption. CNN, ACNN, CNNA, ACNNA, L-CAR, L-histidine, or HH (2 mmol/L)
was incubated with 4-HNE (200 µM) in phosphate-buffered saline (PBS, pH 7.4) at
37°C. The ability of the compounds to capture 4-HNE was evaluated by measuring
4-HNE consumption using high-performance liquid chromatography (CAPCELL PAK
C18 UG80 at 37°C, eluted by acetonitrile/10 mM PBS (pH 7.4) (60/40 (v/v)), flow rate
0.50 ml/min). Captured 4-HNE (%) was calculated as follows:
Captured 4-HNE (%) = 100 –[(AT/A0)×100]
A0: 4-HNE peak area at time 0
AT: 4-HNE peak area at time T
4.5. Cell culture and MTT assay
The rat adrenal pheochromocytoma cell line, PC-12, was maintained in RPNI-1640
(Wako, Osaka, Japan) supplemented with 5% heat-inactivated fetal bovine serum
(Biosera, Nuaille, France) and 10% heat-inactivated horse serum (ThermoFisher
Scientific, Waltham, MA, USA). PC-12 cells were seeded at 2.5 x 105 cells/well in a
24-well plate in 250 µL of the culture medium. After 1 day, various concentrations of
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2.5 µL CNN dimethyl sulfoxide solution were added to the medium for 30 min. The
culture medium containing CNN was then replaced with fresh, CNN-free medium.
Finally, 4-HNE ethanol solution (Merck Millipore, Billerica, MA, USA) was added, and
cells were further incubated for another 1 day before the MTT assay was conducted.
4.6. BCCO
Transient cerebral ischemia was induced by temporary BCCO in Mongolian gerbils.
Briefly, animals were anesthetized with 2% isoflurane in a mixture of 30% oxygen and
70% nitrous oxide. The common carotid arteries were then dissected from their sheaths
via a ventral midline cervical incision and occluded with ligatures for 5 min. After
removing the ligatures, the wound was closed. The rectal temperature was maintained
between 37.5°C and 38.5°C by a self-regulating heat pad during cerebral ischemia. All
procedures were performed accurately under a microscope.
Mongolian gerbils were assigned into five groups: (1) sham-operated group (BCCO was
not induced, n = 6), (2) placebo-treated group (2 ml of distilled water was administered
intraperitoneally 20 min prior to BCCO, n = 12), (3) CNN pre-administration group (2
mL of CNN [20 mg/kg] was intraperitoneally administered 20 min prior to BCCO, n =
12), (4) CNN post-administration group (2 mL of CNN [100 mg/kg] was
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intraperitoneally administered immediately after BCCO, n = 11), (5) HH
post-administration group (2 mL of HH [100 mg/kg] was intraperitoneally administered
immediately after BCCO, n = 7).
4.7. Histopathological evaluation
In order to determine the rate of DND, Mongolian gerbils were perfused with 4%
paraformaldehyde in 0.1 M PBS (pH 7.4) following a heparinized saline flash under
deep anesthesia with isoflurane 7 days after BCCO. The brains were then removed and
post-fixed in the same fixative overnight at 4°C. Brain tissues were embedded in
paraffin, and 4 µm-thick, successive coronal sections were prepared using a microtome.
Brain sections were stained with hematoxylin-eosin, and hippocampal CA1 damage was
evaluated by counting the number of surviving pyramidal neurons within the middle
CA1 subfield at high power using a light microscope. Only neurons with visibly normal
nuclei were counted.
4.8. Immunohistopathological evaluation
For immunohistopathological evaluation, we compared 4-HNE and Hsp70
immunoreactivity in the hippocampal CA1 region of placebo-treated and CNN
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pre-administration groups 24 h after the induction of BCCO. Brain sections were
prepared in the same manner as those for histopathological evaluation. Then, 4-HNE as
an oxidative stress marker was analyzed using antibodies against 4-HNE 1:100 (abcam,
Cat. #: ab48506; Cambridge, UK), and Hsp70 as a neuroprotective marker was analyzed
using antibodies against Hsp70 1:1000 (abcam, Cat. #: ab181606; Cambridge, UK).
All pathological evaluations were performed by two neuropathologists (JM and YS) in
independent readings.
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Acknowledgements
Source of funding
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of
Education, Sports, Science, and Culture of Japan, and by Grants-in-Aid for Challenging
Exploratory Research 16K15143 to M. O. from Japan Society of Promotion of Science.
Disclosure
Declarations of interest: none
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References
[1] M. Singh, A. Kapoor, A. Bhatnagar, Oxidative and reductive metabolism of
lipid-peroxidation derived carbonyls, Chem. Biol. Interact. 234 (2015) 261–273.
[2] H.M. Semchyshyn, Reactive carbonyl species in vivo: generation and dual
biological effects, Sci. World. J. (2014) 417842.
[3] S.W. Hwang, Y.M. Lee, G. Aldini, K.J. Yeum, Targeting Reactive Carbonyl Species
with Natural Sequestering Agents, Molecules. 21 (2016) 280.
[4] G. Vistoli, D. De Maddis, A. Cipak, N. Zarkovic, M. Carini, G. Aldini, Advanced
glycoxidation and lipoxidation end products (AGEs and ALEs): An overview of their
mechanisms of formation, Free. Radic. Res. 47 (2013) 3–27.
[5] T. Urabe, Y. Yamasaki, N. Hattori, M. Yoshikawa, K. Uchida, Y. Mizuno,
Accumulation of 4-hydroxynonenal-modified proteins in hippocampal CA1 pyramidal
neurons precedes delayed neuronal damage in the gerbil brain, Neuroscience. 100
(2000) 241–250.
25

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[6] I. Kruman, A.J. Bruce-Keller, D. Bredesen, G. Waeg, M.P. Mattson, Evidence that
4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis, J. Neurosci. 17
(1997) 5089–5100.
[7] C.H. Lee, K.Y. Yoo, I.K. Hwang, J.H. Choi, O.K. Park, H. Li, et al., Hypothyroid
state does not protect but delays neuronal death in the hippocampal CA1 region
following transient cerebral ischemia: focus on oxidative stress and gliosis, J. Neurosci.
Res. 88 (2010) 2661–2668.
[8] T. Kirino, Delayed neuronal death in the gerbil hippocampus following ischemia,
Brain. Res. 6(239) (1982) 57–69.
[9] W. Zuo, W. Zhang, N. Han, N.H. Chen, Compound IMM-H004, a novel coumarin
derivative, protects against CA1 cell loss and spatial learning impairments resulting
from transient global ischemia, C.N.S. Neurosci. Ther. 21 (2015) 280–288.
[10] A. Heron, H. Pollard, F. Dessi, J. Moreau, F. Lasbennes, Y. Ben-Ari, et al.,
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