DOI: 10.4274/atfm.galenos.2020.35229
RESEARCH ARTICLE / ARAŞTIRMA MAKALESİ
Ankara Üniversitesi Tıp Fakültesi Mecmuası 2020;73(1):42-46
SURGICAL MEDICAL SCIENCES / CERRAHİ TIP BİLİMLERİ
Neuroprotective Effect of Paeonol in the Rat Model of Traumatic
Brain Injury
Rat Travmatik Beyin Hasarı Modelinde Paeonol’ün Nöroprotektif Etkisi
Emine Arık1, Habibullah Dolgun2, Pınar Kuru Bektaşoğlu3,
Özden Çağlar Öztürk3, Levent Gürses2, Bora Gürer3
Julide Ergil1,
Banu Coşkun Yılmaz4,
Çağhan Tönge2,
1University of Health Sciences Turkey, Dışkapı Education and Research Hospital, Clinic of Anaesthesiology and Reanimation, Ankara, Turkey
2University of Health Sciences Turkey, Dışkapı Education and Research Hospital, Clinic of Neurosurgery, Ankara, Turkey.
3University of Health Sciences Turkey, Fatih Sultan Mehmet Education and Research Hospital, Clinic of Neurosurgery, İstanbul, Turkey
4Mersin University School of Medicine, Department of Histology and Embryology, Mersin, Turkey
Abstract
Objectives: Traumatic brain injury (TBI) is a major cause of disability and mortality that induces oxidative stress and apoptosis causing cellular
damage. Several animal models have shown paeonol to be a powerful antioxidant, antiapoptotic, and neuroprotective substance. This study aimed
to investigate possible neuroprotective effects of paeonol in a rat TBI model.
Materials and Methods: Thirty-two male rats were divided into four groups: control, trauma, vehicle, and paeonol groups. Trauma, vehicle, and
paeonol groups were subjected to closed-head, contusive weight-drop injuries. The vehicle (saline) or paeonol (50 mg/kg) was orally administered as
premedication for 15 days. Brain samples were obtained 24 hours after trauma. Histomorphological evaluation of the cerebral cortex was performed
using electron and light microscopy.
Results: Histopathological examination revealed that the TBI-induced cerebral cortex damage was less in the paeonol group.
Conclusion: Paeonol exhibited neuroprotective and anti-edematous effects against TBI.
Key Words: Anti-edema, Neuroprotection, Paeonol, Traumatic Brain Injury
Öz
Amaç: Travmatik beyin hasarı (TBH), oksidatif stres ve hücresel hasara neden olan apoptozu indükleyen temel bir sakatlık ve ölüm nedenidir. Bazı
hayvan modelleri paeonolün güçlü bir antioksidan, antiapoptotik ve nöroprotektif madde olduğunu göstermiştir. Bu çalışma, paeonolün rat TBI
modelinde olası nöroprotektif etkilerini araştırmayı amaçlamıştır.
Gereç ve Yöntemler: Otuz iki erkek rat dört gruba ayrıldı: kontrol, travma, taşıyıcı ve paeonol. Travma, taşıyıcı ve paeonol gruplarında kapalı
kafa travması ağırlık düşürülerek uygulandı. Taşıyıcı (serum fizyolojik) veya paeonol (50 mg/kg) 15 gün boyunca premedikasyon olarak oral
yoldan uygulandı. Beyin örnekleri travmadan 24 saat sonra alındı. Serebral korteksin histomorfolojik değerlendirmesi elektron ve ışık mikroskopisi
kullanılarak yapıldı.
Bulgular: Histopatolojik incelemede, paeonol grubunda TBH kaynaklı serebral korteks hasarının daha az olduğu görüldü.
Sonuç: Paeonol, TBI’ya karşı nöroprotektif ve antiödematöz etkiler sergilemiştir.
Anahtar Kelimeler: Antiödem, Nöroproteksiyon, Paeonol, Travmatik Beyin Hasarı
Address for Correspondence/Yazışma Adresi: Dr. Pınar Kuru Bektaşoğlu, University of Health Sciences Turkey, Fatih Sultan Mehmet Education and Research Hospital,
Clinic of Neurosurgery, İstanbul, Turkey
E-mail: pnr.kuru@gmail.com ORCID: orcid.org/0000-0001-9889-9955
Received/Geliş: 10.01.2020 Accepted/Kabul: 06.05.2020
©️Copyright 2020 Ankara University Faculty of Medicine
Journal of Ankara University Faculty of Medicine is published by Galenos Publishing House.
All content are under CC BY-NC-ND license.
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Ankara Üniversitesi Tıp Fakültesi Mecmuası 2020;73(1)
Arık et al. Neuroprotective Effect of Paeonol in the Rat Model of Traumatic Brain Injury
Introduction
Traumatic brain injury (TBI) is responsible for approximately
one-third of injury-related mortality and is the leading cause
of disability (1). The primary injury occurring during the
trauma causes direct mechanical damage to the neuronal
and surrounding supportive cells and vascular structures. The
secondary injury occurs within minutes after trauma, resulting
in further cell death because of several signaling cascades,
such as oxidative stress, apoptosis, inflammation, ischemia,
mitochondrial dysfunction, and neurotransmitter excitotoxicity
(2,3). Secondary injuries are preventable, although the cause of
functional disability may require hours or years to resolve (4).
The last decade witnessed research regarding several potential
neuroprotective agents; however, none of these agents were
approved for clinical use, except for amantadine sulfate (3,59). After TBI, the reactive oxygen species and pro-inflammatory
cytokines are considered to play crucial roles (7,10). Oxidative
stress also induces apoptosis through increased caspase-3
activity (9,11).
Paeonol (2′-hydroxy-4′-methoxyacetophenone) is a major
phenolic component of the Chinese herbal medicines; Moutan
Cortex of Paeonia suffruticosa Andrews and the root of Paeonia
lactiflora Pall. Paeonol has been shown to exhibit antipyresis,
antiapoptotic, anti-inflammatory, antiallergic, antimicrobial,
antitumor, antidepressant, analgesic, and sedative properties
(12,13). In addition to the above-mentioned activities, paeonol
has also been suggested to have properties of scavenging free
radicals, antioxidation, and anti-platelet aggregation. Paeonol
is proved to have activity in decreasing Ca2+ influx by blocking
L-type Ca2+ channels (13). It prevents microglial activation
and inhibits the activation of several inflammatory signaling
pathways (14,15). Furthermore, chronic treatment with paeonol
inhibits endoplasmic reticulum stress-mediated oxidative stress
(16).
Despite earlier studies on the neuroprotective activity of
paeonol in animal models, its activity in TBI remains unexplored.
The present study investigates the neuroprotective activity of
paeonol in a rat model of TBI for the first time.
Materials and Methods
Experimental Groups
Animal care and all experiments were conducted according
tothe European Parliament and Council directive 2010/63/EU of
September 22, 2010 with regard to the protection of animals
for experimental use. Animal ethics committee permission is
obtained from The Saki Yenilli Animal Care and Use Committee
(0001.01.02). They reviewed and approved all experimental
procedures used in this study. Thirty-two adult male Wistar
albino rats weighing 350-450 g were used. Animals were housed
in an air-conditioned room with 12 h light and dark cycles with
constant temperature (22±2 °C) and relative humidity (65-70%).
Rats were fed standard laboratory chow and had free access to
water.
The rats were randomly assigned to the following four
groups:
1. Control group (n=8): Rats underwent only a skin incision.
Non-traumatized brain samples were obtained 24 h after
surgery. The brain divided into 1-mm3 pieces and stored in
glutaraldehyde for electron and light microscopic examination.
2. Trauma group (n=8): Rats underwent TBI as described
below. Brain samples were obtained 24 h after surgery and were
used for histopathological analysis.
3. Vehicle group (n=8): Rats underwent TBI as described
below and received a 15-day oral dose of the vehicle (0.9%
NaCl, 0.1 mL/100 g). Brain samples were removed 24 h after
injury and were used for histopathological analysis.
4. Paeonol group (n=8): Rats received a 15-day oral dose
of paeonol (50 mg/kg; Sigma-Aldrich, St. Louis, Missouri, USA)
as premedication before TBI. The chosen dose of paeonol was
based on previous studies (13,17). Brain samples were removed
24 h after TBI and were used for histopathological analysis.
Anesthesia and Induction of TBI
The animals were anesthetized using an intraperitoneal
injection of 10-mg/kg xylazine (Rompun, Bayer, Turkey) and
50-mg/kg ketamine (Ketalar, Parke-Davis, Turkey) combination
and allowed to breathe spontaneously. The moderate brain
injury model described by Marmarouet al. (18) and modified
by Ucar et al. (19) was applied for head trauma. The rats were
placed in a prone position on the table and were supported on
a 10-cm foam bed to provide deceleration after the impact. A
midline incision was created on the head, and the coronal and
lambdoid sutures were identified. A metallic disk of 10-mm
diameter and 3-mm thickness was fixed to the cranium using
bone wax between the two sutures in the midline. Trauma was
applied at the point where the disk was placed on the midline.
A lead object weighing 300 g was allowed to fall freely from a
height of 1 m through a copper tube on to the metal disk over
the skull of the rat. After the induction of injury, the metallic
disk was removed, the surgical area was cleaned, and the skin
was sutured. All the animals were decapitated 24 hours after
trauma, and the brains were carefully removed.
Sample Preparation for Electron and Light Microscopy
For transmission electron microscopic evaluation, the brain
tissue samples were fixed with 2.5% glutaraldehyde, postfixed
with 1% osmium tetroxide, dehydrated in a graded alcohol
43
Arık et al. Neuroprotective Effect of Paeonol in the Rat Model of Traumatic Brain Injury
series, cleared with propylene oxide, and embedded in Epon
(EMS, Cat No: 13940).
Ankara Üniversitesi Tıp Fakültesi Mecmuası 2020;73(1)
treated group compared with that in the trauma and vehicle
groups (Figure 5).
Semi-thin sections (2000 nm) were cut using an
ultramicrotome (Leica EM UC7, Leica Microsystems GmbH,
Vienna, Austria) and stained with toluidine blue. These sections
were examined using a light microscope (Olympus BX50) and
photographed (Olympus LC30).
Thin sections (70 nm) were cut using an ultramicrotome and
contrasted with uranyl acetate and lead citrate. These sections
were examined and photographed usinga transmission electron
microscope (JEOL JEM-1011, Jeol Ltd., Tokyo, Japan).
Figure 3: Electron microscopic image of vehicle group. Capillary (C),
intracellular vacuole in the neuronal processes (white arrow head),
intracellular edema and vacuoles in the adjacent to the oligodendrocyte
(O) (white arrow), perivascular edema (asterisk), and synapse (black arrow
head). A X7500, B X7500, C X5000
Results
Electron Microscopic Findings
The control group revealed normal morphological features
of neuron and glial cells, myelin sheath, axon, and neuronal
processes (Figure 1). On examination, the trauma group revealed
significant perivascular edema, with remarkable intracellular
edema and vacuoles observed in the neuronal processes adjacent
to the oligodendrocytes. The axons and myelin sheath showed
degenerative changes (Figure 2). Furthermore, the vehicle group
revealed perivascular edema, with intracellular edema and
vacuoles in neuronal processes (Figure 3). Myelin damage was
observed in the paeonol group, although reduced perivascular
edema was observed in the paeonol group compared with
that in the trauma group. In addition, intracellular edema and
vacuoles in the neuronal processes were decreased (Figure 4).
Light Microscopic Evaluation
Semi-thin sections belonging to study groups were examined
and reduced perivascular edema was observed in the paeonol-
Figure 4: Electron microscopic image of paeonol group. Neuron (N),
Oligodendrocyte (O), Capillary (C), intracellular vacuole in the neuronal
processes (np) (white arrow head), myelin sheath (blackarrow) and
synapse (black arrow head), perivascular edema (asterisk). A X7500, B
X10000, C X6000, D X10000
Figure 1: Electron microscopic image of control group. Normal
morphological apperance of Neuron (N), Oligodendrocyte (O), Capillary
(C), Neuronalprocesses (np). A and B X5000, C X7500
Figure 2: Electron microscopic image of trauma group. Capillary (C),
intracellular vacuole in the neuronal processes (np) (white arrow head),
intracellular edema and vacuoles in the adjacent to the oligodendrocyte
(O) (white arrow), perivascular edema (asterisk), synapse (black arrow
head). A X7500, B X4000, C X5000
44
Figure 5: Light microscopic image, semithin sections. (A) Control group,
(B) Trauma group, (C) Vehicle group, (D) Paeonol group. Neuron (black
arrow head), glial cells (white arrow head), perivascular edema (black
arrow), vessels (v). Toluidine blue, X400
Ankara Üniversitesi Tıp Fakültesi Mecmuası 2020;73(1)
Arık et al. Neuroprotective Effect of Paeonol in the Rat Model of Traumatic Brain Injury
Discussion
TBI is one of the most significant causes of disability and
mortality in young people worldwide (20). Secondary injury, after
the initial mechanical injury during the incident, is triggered
by oxidative stress, apoptosis, inflammation, excitotoxicity,
ischemic processes, and mitochondrial pathways, resulting
in neuronal loss (3). The last decade has witnessed decreased
mortality rates and increased functional survival rates after
TBI because of enhanced knowledge of TBI pathophysiology,
improvement of intensive care services, technological
developments in monitoring, and follow-up of patients (9).
However, despite extensive research regarding neuroprotective
agents, no clinically effective pharmacological treatment has
been developed for TBI (3,6,7,9,11). Consequently, TBI treatment
has garnered interest and extensive research is underway to
develop a possible therapeutic agent.
Paeonol is a major phenolic component of Moutan bark, the
root bark of Paeoniasuffruticosa Andrews (Paeoniaceae) that
is a traditional Chinese herbal medicine (21). It is known for
its broad range of therapeutic properties probably because of
its free radical scavenging and anti-inflammatory properties,
including antiproliferative, antiplatelet aggregation, and
neuroprotective activities (22,23). It can cross the blood–brain
barrier because of its small molecular weight (24).
In the current study, the histopathological assessment of
the brain tissues was performed at both light microscopic and
ultrastructural levels. The brain morphology was normal in the
control group, where as significant perivascular edema was
observed in the trauma and vehicle groups. Intracellular edema
is the first sign of cellular injury. Swelling, vacuolar changes, and
lysis of some organelles are significant signs of acute cellular
injury. In this study, the results of electron microscopic analysis
also confirmed TBI related injury at cellular level. Furthermore,
the intracellular edema and vacuoles that observed in the
trauma and vehicle groups were observed to be decreased in the
paeonol group.
Wu et al. (12) showed that paeonol could protect oxygenglucose-deprived hippocampal neurons by preventing
excitotoxicity through NMDA receptors in cell culture. Zhong
et al. (24) indicated that the neuroprotective effect of paeonol
could be because of its free radical scavenging and antioxidant
properties. It could additionally protect Na+/K+-ATPase activity
and preserve energy metabolism (24). Recent studies conducted
in cerebral ischemia animal models reported that paeonol
pretreatment decreased cerebral edema and infarct volume
besides preserving the blood–brain barrier and inhibiting
microglial activation (23,25). Zhao et al. (26) investigated
the effects of paeonol treatment after cerebral ischemia and
reported that paeonol suppresses microglial activation and
astrocyte proliferation and exhibits a neuroprotective effect.
In addition, Liao et al. (27) showed neuroprotective and antiinflammatory activities of paeonol in a cerebral ischemia and
reperfusion model.
This study had some limitations. First, the use of paeonol
as a pretreatment could decrease its practical application,
particularly in emergency trauma situations. If results for
inflammatory and other biochemical biomarkers, quantitative
light and electron microscopy, and functional outcome scores
had been obtained, the effects of paeonol could have been
interpreted more mechanistically. Future studies using TBI
animal models with an increased number of animals per group
and different dosage regimens at different time periods can be
conducted to understand the effectiveness of paeonol better.
Furthermore, other TBI animal models (i.e., repetitive TBI models)
can be studied.
Conclusion
This study is the first to evaluate the neuroprotective
properties of paeonol in TBI. Paeonol has been shown to be
effective in preventing neural damage secondary to TBI. After
further experimental and clinical studies, paeonol may be
approved for the TBI treatment.
Ethics
Ethics Committee Approval: Animal care and all
experiments were conducted according tothe European
Parliament and Council directive 2010/63/EU of September 22,
2010 with regard to the protection of animals for experimental
use. Animal ethics committee permission is obtained from The
Saki Yenilli Animal Care and Use Committee (0001.01.02). They
reviewed and approved all experimental procedures used in this
study.
Informed Consent: Due to the fact that this study is
experiment study, informed consent was not obtained.
Peer-review: Internally and externally peer-reviewed.
Authorship Contributions
Concept: E.A., H.D., P.K.B., J.E., B.C.Y., Ç.T., Ç.Ö.Ö., L.G., B.G.,
Design: E.A., H.D., P.K.B., J.E., B.C.Y., Ç.T., Ç.Ö.Ö., L.G., B.G., Data
Collection or Processing: E.A., H.D., P.K.B., J.E., B.C.Y., Ç.T., Ç.Ö.Ö.,
L.G., B.G., Analysis or Interpretation: E.A., H.D., P.K.B., J.E., B.C.Y.,
Ç.T., Ç.Ö.Ö., L.G., B.G., Literature Search: E.A., H.D., P.K.B., J.E.,
B.C.Y., Ç.T., Ç.Ö.Ö., L.G., B.G., Writing: P.K.B.
Conflict of Interest: No conflict of interest was declared by
the authors.
Financial Disclosure: The authors declared that this study
received no financial support.
45
Arık et al. Neuroprotective Effect of Paeonol in the Rat Model of Traumatic Brain Injury
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