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 (3,5-9). 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 series, cleared with propylene oxide, and embedded in Epon (EMS, Cat No: 13940).
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).
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-treated group compared with that in the trauma and vehicle groups (Figure 5).
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 oxygen-glucose-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 anti-inflammatory 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.
