Effects of thymol on amyloid-β-induced impairments in hippocampal synaptic plasticity in rats fed a high-fat diet

Brain Research Bulletin 137 (2018) 338–350

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Research report

Effects of thymol on amyloid-β-induced impairments in hippocampal synaptic plasticity in rats fed a high-fat diet

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Masoumeh Asadbegia,b, Alireza Komakia, ,1, Iraj Salehia, Parichehreh Yaghmaeib, Azadeh Ebrahim-Habibic,d, Siamak Shahidia, Abdolrahman Sarihia, Sara Soleimani Asla, Zoleikha Golipoora a

Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran c Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran d Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b

A R T I C L E I N F O

A B S T R A C T

Keywords: Thymol Alzheimer’s disease β-Amyloid Long term potentiation Hippocampus High-fat diet

Obesity and a high-fat diet (HFD) are known to increase the incidence of Alzheimer’s disease (AD). Oxidative stress, a major risk factor for AD, is increased with HFD consumption. Thymol (Thy) has antioxidant properties. Therefore, in the present study, we examined the protective and therapeutic effects of Thy on amyloid-β (Aβ)induced impairments in the hippocampal synaptic plasticity of HFD-fed rats. In this study, 72 adult male Wistar rats were randomly assigned to 9 groups (n = 8 rats/group): Group 1 (control; standard diet); Group 2: Control + phosphate-buffered saline (PBS) + Oil (Thy vehicle); Group 3 (HFD + PBS); Group 4: (HFD + Aβ); Group 5: Control + PBS + Thy; Group 6: (HFD + Aβ + Oil); Group 7: Control + Aβ + Thy; Group 8: HFD + PBS + Thy; Group 9: (HFD + Aβ + Thy). After stereotaxic surgery, the field potentials were recorded after the implantation of the recording and stimulating electrodes in the dentate gyrus (DG) and perforant pathway, respectively. Following high-frequency stimulation, the long-term potentiation (LTP) of the population spike (PS) amplitude and the slope of the excitatory postsynaptic potentials (EPSPs) were measured in the DG. The HFD rats that received Aβ exhibited a significant decrease in their EPSP slope and PS amplitude as compared to the control group. In contrast, Thy administration in the HFD + Aβ rats reduced the decrease in the EPSP slope and PS amplitude. Thy decreased the Aβ-induced LTP impairments in HFD rats. The HFD significantly increased serum malondialdehyde levels and decreased total antioxidant capacity and total glutathione levels; whereas, Thy supplementation significantly reversed these parameters. Therefore, these results suggest that Thy, a natural antioxidant, can be therapeutic against high risk factors for AD, such as HFD.

1. Introduction Overweight and obesity are estimated to affect over 2 billion people worldwide (Ng et al., 2014), which has resulted in an enormous strain on healthcare systems (Petrov et al., 2015). Human obesity is associated with the consumption of high-fat diets (HFDs) (Moy and McNay, 2013). Although the relationship between obesity and its adverse effects on the brain remains unclear, studies have suggested that obesity and body fat deposition play an important role in the pathogenesis of certain brainrelated disorders (Kim et al., 2015). Furthermore, increasing evidence suggests that obesity and HFD can also cause long-term memory loss, neuronal damage, and cognitive impairment (Farr et al., 2008; Kim et al., 2015; Moy and McNay, 2013).

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Alzheimer’s disease (AD) is the most common form of dementia and is a significant worldwide health problem. AD is one of the most common neurodegenerative diseases, and is characterized by progressive functional disturbances in cognition and memory (Peng et al., 2013). AD has characteristic molecular and biochemical abnormalities, including cell loss, amyloid-β (Aβ) deposits, chronic oxidative stress, and DNA damage (Suzanne and Wands, 2008). There are several factors that can increase AD risk, including diabetes, stroke, atherosclerosis, obesity and HFD consumption (Knight et al., 2014). Moreover, in transgenic mouse models, high-fat diets increase the deposition of Aβ peptides (Levin-Allerhand et al., 2002). HFD in animal models of AD is associated with an increased accumulation of the toxic Aβ peptide and impaired behavior (Asadbegi et al., 2017; Barron et al., 2013; Julien

Corresponding author at: Department of Physiology, School of Medicine, Hamadan University of Medical Sciences, Shahid Fahmideh Street, Hamadan, 65178/518, Iran. E-mail address: [email protected] (A. Komaki). URL: umsha.ac.ir.

https://doi.org/10.1016/j.brainresbull.2018.01.008 Received 12 March 2017; Received in revised form 1 January 2018; Accepted 8 January 2018 Available online 13 January 2018 0361-9230/ © 2018 Elsevier Inc. All rights reserved.

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2.2. Diet composition

et al., 2010). The balance between oxidation and anti-oxidation is thought to be critical in maintaining a healthy biological system (Bouayed and Bohn, 2010; Khodamoradi et al., 2015). Oxidative stress refers to an imbalance between the production of reactive oxygen species and the antioxidant defense system, which buffers the system from oxidative damage. Oxidative stress is implicated in the pathogenesis of several diseases (Betteridge, 2000; Yilmaz et al., 2005). Diets that are enriched in natural antioxidants have beneficial effects on oxidative stress, blood pressure, and serum lipid composition (Vargas-Robles et al., 2015). Obesity and AD markers are associated with oxidative stress and mitochondrial dysfunction, which are mechanisms that can lead to neurodegeneration (Nuzzo et al., 2015). In AD pathogenesis, oxidative stress may be a major risk factor for the underlying causal mechanisms of AD (Holmes, 2013; Markesbery, 1997; Morris and Tangney, 2014). Thymol (2-isopropyl-5-methylphenol) (Thy) is a natural phenolic compound that is present in various plants, such as thyme (Lamiaceae) and Zataria (Hosseinimehr et al., 2011; Sajed et al., 2013; Schmidt et al., 2012). Several of the biological properties of Thy are mediated through its antioxidant effects, including its protective effects against toxicity (Al-Malki, 2010; Alam et al., 1999; Arab et al.; Archana et al., 2011). The significant antioxidant activities of Thy may be helpful in preventing the progress of various oxidative stress-related diseases, justifying its use in traditional medicine (Aman et al.). Recent studies have confirmed that antioxidant-rich foods play a vital role in the disease prevention of neurodegenerative diseases, including AD (Esmaeili and Khodadadi, 2012; Gerber et al., 2002). In the present study we examined the protective and therapeutic effects of Thy on the Aβ-induced long-term potentiation (LTP) impairments in HFDfed rats. LTP is a type of synaptic activity that has been thoroughly studied in the hippocampus, and is thought to be the neural correlate of learning and memory (Hölscher, 1999; Tahmasebi et al., 2015). If Thy is protective against AD-related impairments, then natural therapeutic agents based on the structure of Thy could be used to protect against oxidative stress-related illnesses, such as AD.

Rats were fed with standard rat food pellets for 7 days in order to recover from transportation and then were divided into nine groups. A standard laboratory rodent chow diet was used for the control diet (Asadbegi et al., 2016b; Belabed et al., 2006; Mobley et al., 2015; Sant’Diniz et al., 2005; Willy et al., 1995). A standard laboratory rodent chow diet was used for the control diet. The control diet was composed of 23% protein, 47% carbohydrate, 5% lipids, 5% cellulose, 20% water, and vitamins and minerals with a caloric density of approximately 3.0 kcal/g (Karamian et al., 2015; Willy et al., 1995). The standardized HFD utilized in this experiment consisted of the subsequent hypercaloric constituents: 15 g of laboratory rat chow, ten g of cooked ground nuts, ten g of milk chocolate, and five g of sugar cookies (de Melo et al., 2010; Estadella et al., 2004; Karamian et al., 2015; Karimi et al., 2015; Komaki et al., 2015). These ingredients were ground and prepared into pellets that contained 20% protein, 48% carbohydrates, 20% lipids, 4% cellulose, and 5% vitamins and minerals by weight. To avoid the autooxidation of the fat components, the food was stored at 20 °C (de Melo et al., 2010; Karamian et al., 2015; Karimi et al., 2015; Komaki et al., 2015). 2.3. Experimental design The animals acclimated for seven days before their use in the experiments. The rats were at random assigned to one of the following groups (n = 8 rats/group): Group 1 (Control; standard lab chow without disruption); Group 2 (Control + phosphate-buffered saline (PBS) + oil; 8 weeks standard lab chow then PBS, followed by sunflower oil by daily oral gavage for 4 weeks); Group 3 (HFD + PBS; consumed a HFD for eight weeks and then PBS); Group 4: (HFD + Aβ; consumed HFD for 8 weeks, followed by Aβ1–42 injections); Group 5 (Control + PBS + Thy; consumed standard lab chow for 8 weeks, followed by PBS injections; after recovery, Thy (30 mg/kg in sunflower oil) was administered by daily oral gavage for 4 weeks); Group 6 (HFD + Aβ + oil; consumed HFD for 8 weeks, followed by Aβ1–42 injections; after recovery, sunflower oil was administered by daily oral gavage for 4 weeks). Group 7 (Control + Aβ + Thy); Group 8 (HFD + PBS + Thy); and Group 9 (HFD + Aβ + Thy; consumed HFD for 8 weeks, followed by Aβ1–42 injections; after recovery, Thy (30 mg/ kg in sunflower oil) was administered by daily oral gavage for 4 weeks). This dose (30 mg/kg) was chosen in line with the previous reports (Du et al., 2016; Ribeiro et al., 2016). Experimental timeline is shown in Fig. 1.

2. Materials and methods 2.1. Animals We used 72 male Wistar laboratory rats, 8–9 weeks old, weighing 120 ± 5 g (Pasteur Institute of Iran, Tehran, Iran). The animals were housed in an air-conditioned room at 22 ± 2 °C under 12 h/12 h light/ dark cycle (light turned on at 07:00 and turned off at 19:00). Four rats were kept in each cage. Water and food were available ad libitum. All of the experiments and animal care methods were confirmed by the Veterinary Ethics Board of the Hamadan University of Medical Science and carried out according to Guidelines of the National Institutes of Health on the principles of laboratory animal care (NIH Publication 8023, 1996).

2.4. Aβ injections and surgery Following 8 weeks of controlled diets in each experimental group, to generate an AD model, the rats were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) and transferred to a stereotaxic device (Stoelting Co., Wood Dale, IL, USA). Aβ1–42 (100 μg; Tocris Bioscience, Bristol, UK) was dissolved in 100 μL of PBS (vehicle

Fig. 1. Experimental timeline. Timeline: Following 8 weeks of standard diet or HFD in experimental groups, to generate an AD model, rats were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) and transferred to a stereotaxic device. The Aβ solution (2 μL) was injected into the dentate gyrus bilaterally at a rate of 1 μL/2 min. After recovery, thymol (30 mg/kg in sunflower oil) was administered by a daily oral gavage for a period of 4 weeks. Then, the rats were anesthetized with an IP injection of urethane and placed in a stereotaxic device for surgical procedure and electrophysiological recording. Once a stable baseline of responses was obtained for at least 20 min, LTP was elicited employing a high-frequency stimulation protocol. At the end of the experiments the biochemical parameters and the biomarkers levels of oxidative stress were determined after serum analysis.

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Fig. 2. A: Exemplary photomicrograph illustrating places of stimulating and recording electrodes tips (arrowheads) in a hippocampus sagittal section. Stimulating and recording electrode traces can be seen at the right and left sides, respectively (arrows). Scale bar: 0.5 mm. B: Schematic drawing of several accepted injection sites in the experiments.

insertion as needed) (Asadbegi et al., 2016b; Karamian et al., 2015; Salehi et al., 2015; Tahmasebi et al., 2015). A heating pad was used to maintain the body temperature of animals. After the incision of the skin and determining the position of the DG according to the Paxinos and Watson atlas for the rat brain (Paxinos and Watson, 2006), small burr holes were drilled into the skull. Recording and stimulating electrodes were positioned in the DG (AP: −3.8 mm from the bregma; ML: 2.3 mm from the midline; DV: 2.7–3.2 mm from the surface of the skull) and perforant pathway (PP) (AP: −8.1 mm from the bregma; ML: 4.3 mm from the midline; DV: 3.2 mm from the surface of the skull). Optimal ventral placement was achieved through electrophysiological monitoring of the response evoked in the DG following single-pulse PP stimulation. The electrodes were lowered very slowly (0.2 mm/min) from the cortex to the hippocampus, in order to minimize trauma to the brain tissue.

solution) and incubated at 37 °C for 7 days prior to use. This process resulted in the development of amyloid fibrils, which are toxic in the nervous system (Lorenzo and Yankner, 1994; Yaghmaei et al., 2013). The injections were performed with a 5-μL microsyringe (Hamilton Laboratory Products, Reno, NV, USA). The coordinates for the dentate gyrus (DG) were −3.6 mm posterior, ± 2.3 mm lateral, and 3 mm dorsal (Asadbegi et al., 2016b; Paxinos et al., 1985) relative to bregma and with the stereotaxic arm at 0°. The Aβ solution (2 μL) was injected into the region bilaterally at a rate of over 1 μL/2 min. The cannula was left in place for 2 min after each injection to allow for diffusion. The sham-operated rats received a vehicle solution (Asadbegi et al., 2016a,b). 2.5. Surgical procedures, electrophysiological recordings, and LTP induction Rats were anesthetized with intraperitoneally administered urethane (ethyl carbamate; Sigma, USA; 1.5 g/kg with supplementary 340

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2.6. HFS for LTP induction

2.8. Plasma measurements

An input/output (I/O) response curve was constructed by varying the intensity of single pulse stimulations and averaging five responses per intensity. The stimulus intensity that evoked a mean field potential equal to 50% of the maximal response was used for all subsequent stimulations. After the determination of I/O curves, single stimuli (duration was 0.2 ms) were applied every 10 s for at least 30 min, and the responses were monitored. Once a stable baseline was obtained for at least 20 min, LTP was induced using a 400-Hz HFS protocol (10 bursts of 20 stimuli, 0.2 ms stimulus duration, 10 s interburst interval) at a stimulus intensity that evoked a PS amplitude that was approximately 50% of the maximal response. PS was recorded 5, 30, and 60 min after HFS in order to determine possible changes in the synaptic response of DG neurons. For each time-point, 10 consecutive evoked responses were averaged at 10 s stimulus intervals (Asadbegi et al., 2016b; Karamian et al., 2015; Karimi et al., 2015; Salehi et al., 2015; Tahmasebi et al., 2015; Taube and Schwartzkroin, 1988). For stimulations, the parameters of the stimuli were defined using our own software (eTrace, www.sciencebeam.com) and were sent to an invariable current isolator system (A365, WPI, Inc. USA) via a data acquisition device prior to transfer to the PP through the stimulus electrode. The induced field potential response from the DG was passed through a preamplifier, amplified (1000×) (DAM 80, WPI, Inc. Sarasota, FL, USA), and filtered (1 Hz–3 kHz). This response was digitized at a selection rate of 10 kHz and was noticeable on a PC. These data were saved to a computer for future offline analysis (Asadbegi et al., 2016b; Karamian et al., 2015; Karimi et al., 2015; Nazari et al., 2016a; Salehi et al., 2015; Tahmasebi et al., 2015).

At the completion of the experiment, 5 mL of blood samples were taken from the portal vein by cardiac puncture and transferred into heparinized tubes and centrifuged at 3000 rpm for 10 min at 4 °C. The plasma was utilized to measurement cholesterol, very-low-density lipoprotein (VLDL), low-density lipoproteins (LDL), and triglycerides (TG). 2.9. Biochemical parameters Also, at the end of the experiments the biochemical parameters and the biomarkers levels of oxidative stress were determined in the serum. Serum measurements were performed for total antioxidant capacity (TAC), total oxidant status (TOS), malonedialdehyde (MDA), and total thiol group (TTG). Protein determination: The protein levels were spectrophotometrically estimated by the method of Bradford, using bovine serum albumin as a standard. Biochemical parameters were adjusted based on total protein content (Bradford, 1976). 2.9.1. Measurement of TAC TAC in serum samples was assessed using ferric reducing antioxidant power assay (FRAP) (Benzie and Strain, 1999). This procedure involves the reduction of ferric tripyridyltriazine (Fe III-TPTZ) to a blue colored Fe II-TPTZ by biological antioxidants. The change of absorbance at 600 nm of the sample was compared with the change of absorbance of a known standard (FeSO4 7H2O) (Salehi et al., 2015). 2.9.2. Measurement of TOS The oxidation of ferrous ion to ferric ion accompanied with a number of oxidant species in acidic pH was used for the measurement of TOS in serum. The ferric ion was determined by xylenol orange (Erel, 2005). Briefly, 225 μL of reagent 1 (150 μM xylenol orange, 140 mM NaCl, and 1.35 M glycerol in 25 mM H2SO4 solution, pH 1.75) was mixed with 35 μL of sample. The absorbance of each sample was read using a spectrophotometer at 560 nm (sample blank) (Karamian et al., 2015).

2.7. Measurements of the evoked potentials The evoked field potential in the DG has 2 components, population spike (PS) and field excitatory postsynaptic potential (fEPSP) (Fig. 2A). During electrophysiological recording, changes in PS amplitude and fEPSP slope were measured. The amplitude of PS was measured from the peak of the first positive deflection of the evoked potential to the peak of the following negative potential. The fEPSP slope function was measured as the slope of the line joining the onset of the early positive deviation of the stimulated potential with the top of the second positive deviation of the stimulated potential. Measurements of the slope of the fEPSP were taken between 20 and 80% of the peak amplitude (Karamian et al., 2015; Karimi et al., 2015; Komaki et al., 2017; Nazari et al., 2016b; Salehi et al., 2015; Tahmasebi et al., 2016). Two components of LTP can be distinguished using extracellular recording electrodes. A synaptic component, reflected in the potentiation of the fEPSP, results in enhanced excitatory input to a neuronal pool and concomitantly increases the number of synchronously discharging cells to produce a larger PS (Chavez-Noriega et al., 1989). The slope of the fEPSP is a measure of synaptic strength at PP- granule cell (GC) synapses. If the stimulation intensity reaches the threshold for GC action potentials, fEPSPs are accompanied by a PS (Andersen et al., 1971). Different mechanisms may underlie the two components of LTP (Chavez-Noriega et al., 1989). The amplitude of the PS reflects the number and synchrony of GC discharges (Andersen et al., 1971). It has been shown that tetanization of the PP was able to induce LTP in the DG in both measured parameters—the fEPSP and the PS (Frey and Frey, 2009). Parallel increase of the fEPSP slope and the PS occurs only after concurrent pre- and postsynaptic activation (Bliss and Collingridge, 1993). For a better characterization of the used stimulus parameters and the inter-comparison (standardization) of used stimulation intensities between different animals, the recording of both, the EPSP as well as the spike is useful (Frey and Frey, 2009).

2.9.3. Measurement of MDA The generation of MDA, which was used to evaluate lipid peroxidation, was determined by reaction with thiobarbituric acid (Kamal et al., 1989). Briefly, the lipid peroxidation products (MDA) were measured by mixing 1.0 mL of 20% trichloroacetic acid and 1.0 mL of 1% thiobarbituric acid reactive substances with 100 μL of the supernatant and incubating the solution at 100 °C for 80 min. After cooling the solution on ice, it was centrifuged at 3000 rpm for 20 min, and the absorbance of the supernatant was read at 532 nm (Ganji et al., 2017a,b; Salehi et al., 2015). 2.9.4. Measurement of the TTG level of the serum Total serum thiol concentration or sulfhydryl groups (thiol) were measured using the methods described originally by Ellman (1959) and modified by Hu (1994). After manuel spectrophotometric optimization processes the method was applied to an automated analyzer. Sample and reagent 1 were mixed. After 90 s, reagent 2 was added. Here, thiols interact with 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB), forming a highly colored anion (5-thio-2-nitrobenzoic acid) with a maximum peak at 412 nm (Eren et al., 2015). 2.10. Histology Following ending of study, the stimulating and recording locations in the hippocampus were verified histologically from brain slices. At the end of study, animals were deeply anesthetized by urethane and perfused through the heart with formol–saline (Komaki and Esteky, 2005; 341

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The serum concentrations of TAC, TOS, MDA, and TTG for all groups are shown in Fig. 4. The mean TAC, expressed as mean concentration ( ± SEM) in plasma, from the HFD + Aβ group (18.99 ± 6.664 μmol/mg protein) were significantly lower than that from the control group (109 ± 10.35 μmol/mg protein; p = 0.0021) (Fig. 4A). The mean level of TTG in the plasma of the HFD + Aβ group (13.03 ± 3.775 μmol/mg protein) was significantly lower than the control (43.75 ± 6.667 μmol/mg protein; p = 0.031) group (Fig. 4B). Moreover, MDA levels in the plasma of the HFD + Aβ group (3.095 ± 0.4374 μmol/mg protein) were significantly higher than those in the control group (1.461 ± 0.098; p = 0.0273) (Fig. 4C). In addition, the plasma concentration of TOS was significantly higher in the HFD + Aβ group (0.998 ± 0.0693) than in the control group (0.1628 ± 0.04769; p = 0.000) (Fig. 4D). The control + PBS + Thy (189.6 + 16.24; p = 0.0056) and control + Aβ + Thy (204.4 ± 9.029; p = 0.0012) groups showed a significant increase in TAC compared to the control (109 ± 10.35) group. In addition, control + Aβ + Thy (204.4 ± 9.029; p = 0.000) and HFD + Aβ + Thy (123.7 ± 12.91; p = 0.0020) groups showed a significant increase in TAC compared to the HFD + Aβ (18.99 ± 6.664) group. Also, this substance could increase mean level 342

Control

Table 1 The initial and final body weight of control and experimental groups of rats.

3.3. Effects of Thymol on TAC, TOS, MDA and TTG of AD (Aβ-injected) rats fed a HFD

Control + PBS + Oil

Control+ PBS + Thy

A considerable difference was shown in the quantity of cholesterol, VLDL, and TG between the control group compared to the HFD group (n = 8; p = 0.000) (Fig. 3). The HFD increased TG (Fig. 3A), VLDL (Fig. 3C) and cholesterol (Fig. 3D) levels in the HFD group. There was no significant difference in the level of LDL (Fig. 3B), among the groups (n = 8; p = 0.445). A significant difference was observed in the level of TG (n = 8; p = 0.000), VLDL (n = 8; p = 0.000) and cholesterol (n = 8; p = 0.000027) between the HFD + Aβ + thy and HFD + Aβ groups.

123.1 ± 1.231 363.8 ± 3.663*** 124.2 ± 1.184 374.2 ± 7.629*** 126.9 ± 1.945 364.7 ± 4.412***

*** P < 0.001 significant difference in compare with control. Data are presented as the mean ± standard error of the mean (SEM). The mean difference is significant at the 0.05 level. n = 8/group.

Control + Aβ + Thy

3.2. Effects of Thymol on the plasma parameters of AD (Aβ-injected) rats fed a HFD

123.3 ± 1.243 362.7 ± 6.587***

HFD + PBS

The body weights were monitored in all groups throughout the experiment. The initial, and finishing weights are revealed in Table 1. The body load at the onset of the alimentary feeding regimen did not significantly differ among the groups (n = 8; p = 0.131). The final body weights at the end of the study significantly differed between the groups. All rats in the HFD groups had significantly higher body weights compared to the weight of the other groups (n = 8; p = 0.000).

128.6 ± 1.688 289.3 ± 5.027

HFD + PBS + Thy

3.1. The effects of Thy treatment on the body weight of AD (Aβ-injected) rats fed a HFD

126.1 ± 1.299 279.7 ± 6.217

3. Results

124.9 ± 1.503 291 ± 6.241

HFD + Aβ

Data were analyzed using the two-way repeated measures ANOVA test followed by Tukey’s test with PASW Statistics 22 (IBM Corporation, Armonk, NY, USA). The data are expressed as mean ± standard error of the mean (SEM). A value of p < 0.05 was considered as significant.

126.5 ± 1.174 285.3 ± 5.561

2.11. Data analysis

Body weight (g) (Primary) Body weight (g) (Last)

HFD + Aβ+ Thy

HFD + Aβ + Oil

Komaki et al., 2013). Coronal brain slices were cut at 50 μm and colored with hematoxylin-eosin for histological investigation and confirmation of electrode tip location by a researcher blinded to the experimental results (Nazari et al., 2016a,b) (Fig. 2). To evaluate amyloid plaques, brain sections were stained with thioflavin S (Asadbegi et al., 2017). Staining results were observed with a fluorescent microscope. In this methods, 8 photomicrographs were prepared from each sample whose amyloid plaques were counted with Image tool software.

123.6 ± 1.349 369.1 ± 3.715***

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Fig. 3. The effects of Thy administration along with HFD on serum levels of TG (A), LDL (B), VLDL (C) and Cholesterol (D). Each column and bar represents mean ± SEM. *p < 0.05, ** p < 0.01 and *** p < 0.001with respect to the control group. ### p < 0.001 with respect to the HFD + Aβ group.

Fig. 4. Plasma parameters of malondialdehyde (MDA), total antioxidant status (TAC), total thiol groups (TTG), and total oxidant status (TOS) of rats in all; n = 8 per group. Data are expressed as mean ± standard error of the mean (SEM) (μmol/mg protein). *p < 0.05, ** p < 0.01 and *** p < 0.001 compared to control group. # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to HFD + Aβ group. & p < 0.05, && p < 0.01 and &&& p < 0.001 compared to HFD + PBS group.

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Fig. 5. Representative sample of input/output trace response (A), sample of input/output curve of PS amplitude (B) in dentate gyrus following PP stimulation.

of TTG in HFD + Aβ + Thy (52.08 ± 12.61 μmol/mg protein; p = 0.0047) group when compared with HFD + Aβ (13.03 ± 3.775) group. Additionally, the control + Aβ + Thy (0.1959 ± 0.07286) group showed a significant decrease in TOS compared to the

HFD + PBS (0.6856 ± 0.1022; p = 0.0021) and HFD + Aβ (0.998 ± 0.0693; p = 0.000) groups. Besides, thymol could decrease the mean level of MDA in control + PBS + Thy (1.106 ± 0.097; p = 0.0162) and HFD + PBS + Thy (1.17 ± 0.090; p = 0.0207) 344

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Fig. 6. The excitatory postsynaptic potential (EPSP) slope and population spike (PS) amplitude of representative sample traces of field potential were recorded in the lateral perforant pathway-dentate gyrus (PP-DG) synapses of a control rat. The arrows indicate PSs and the slope of the EPSP (A). Representative sample traces of evoked field potential in the DG recorded prior to and after high frequency stimulation (HFS) in all groups (B).

groups when compared with HFD + Aβ (3.095 ± 0.4374) group. In summary, we found a lower TAC and TTG, but, higher levels of MDA and TOS in the HFD + Aβ group, and a higher TAC and TTG, but, lower levels of MDA and TOS, in the thymol treated groups, when compared with the control and vehicle groups.

(103.4 ± 3.423%; n = 8; p = 0.000047) rats. However, the 4 weeks of Thy treatment significantly increased the EPSP slope in the Control + Aβ + Thy (126.9 ± 9.277%; n = 8; p = 0.000019) and HFD + Aβ + Thy rats (117.3 ± 5.828%; n = 8; p = 0.000179), compared to that of the HFD + Aβ rats (98.32 ± 1.676%; n = 8).

3.4. The effects of Thy on the EPSP slopes of DG granular cells in AD rats fed a HFD

3.5. Effects of Thy on the PS amplitudes of DG granular cells in AD rats fed a HFD

The input/output responses (Fig. 5) and field potential recordings (Fig. 6) were obtained in the DG granular cells following the stimulation of the PP. LTP was induced in the DG by HFS of the PP. The effects of the Thy treatment after Aβ injections on the EPSP slopes and PS levels of the AD rats fed a HFD are revealed in Figs. 7 and 8, respectively. The EPSP slope was 117.5 ± 6.116% of the baseline in the control group rats. The EPSP slope in the Control + PBS + Oil (111.6 ± 5.127%; n = 8; p = 0.959), and HFD + PBS (114.5 ± 5.193%; n = 8; p = 0.999) groups did not significantly differ from the control group’s slope. The intrahippocampal injection of Aβ significantly decreased the hippocampal EPSP slopes in the HFD + Aβ (98.32 ± 1.676%; n = 8; p = 0.000049), and HFD + Aβ + Oil

In control rats, the PS amplitude was 215.9 ± 38.76% of the baseline response. The PS amplitude in the HFD + Aβ (108.3 ± 3.225%; n = 8; p = 0.000), and HFD + Aβ + Oil (129.7 ± 9.085%; n = 8; p = 0.000) was significantly decreased compared to in the control (215.9 ± 38.76%; n = 8) group. The PS amplitudes in the HFD + Aβ (108.3 ± 3.225%; n = 8; p = 0.000002), and HFD + Aβ + Oil (129.7 ± 9.085%; n = 8; p = 0.000012) were significantly decreased compared to in the HFD + PBS (193.9 ± 31.32%; n = 8) group. The PS amplitude in the Control + Aβ + Thy (221.4 ± 41%; n = 8; p = 0.00004) and HFD + Aβ + Thy (187.4 ± 7.439%; n = 8; p = 0.001) groups were significantly increased compared to the HFD + Aβ group (108.3 ± 3.225; n = 8). 345

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Fig. 6. (continued)

distinguished as brighter spots (Fig. 9). The intrahippocampal injection of Aβ significantly increased the number of plaques in the brain tissue (p = 0.000 compared with control). Thymol consumption caused a significant reduction of the plaques number (p = 0.000).

3.6. Histological studies Amyloid plaques were investigated by Thioflavin S staining, which results into a fluorescence in amyloid plaques that could be 346

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Fig. 7. The effects of Thy administration along with HFD on the excitatory postsynaptic potential (EPSP) slope in the dentate gyrus (DG) using 400 Hz tetanic stimulation of AD-induced rats. Long-term potentiation (LTP) of the EPSP slope in DG synapses was considerably different between the groups. The data are plotted as the average percentage change from baseline responses. Data are expressed as mean% of the baseline ± SEM. Comparisons were made using a two-way repeated measures analysis of variance followed by post hoc Tukey’s multiple comparisons test. *p < 0.05, ** p < 0.01 and *** p < 0.001 compared to control group. # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to HFD + Aβ group. & p < 0.05, && p < 0.01 and && & p < 0.001 compared to HFD + PBS group.

4. Discussion

neuropathology and cognitive deficits in AD mouse models (Knight et al., 2014). Accordingly, it has been reported that maternal over-nutrition due to HFD during pregnancy and lactation in an animal model of AD accelerated AD disease pathology and impaired behavior in their offspring (Martin et al., 2014). It has been revealed that a HFD and Aβ injection decreased LTP induction in rats (Asadbegi et al., 2016b). In line with this, the numbers of pyramidal neurons in rat hippocampal CA1 subfield were significantly decreased in Aβ-injected rats fed an HFD (Asadbegi et al., 2016a). Impaired spatial memory and hippocampal synaptic plasticity has also been reported in genetically obese animal models (Gerges et al., 2003; Hwang et al., 2010; Li et al., 2002).

Here, we investigated whether Thy treatment can attenuate Aβ-induced synaptic plasticity impairments in HFD-fed rats in vivo. In the present study, we found that HFD rats that received Aβ and Thy had less of a decrease in their EPSP slopes and PS amplitudes compared to these measures in HFD + Aβ rats. These data suggest that Thy attenuated the HFD + Aβ-induced impairments in LTP induction in the PP-DG pathway. HFD has multiple associations with cognitive impairment (Komaki et al., 2015; Mody et al., 2011). HFD can also increase the disease

Fig. 8. The effects of Thy administration along with HFD on the magnitude of the population spike (PS) in the dentate gyrus (DG) using 400 Hz tetanic stimulation. Long-term potentiation (LTP) of PS amplitudes in DG granular cells in the hippocampus was significantly different between the groups. The data are plotted as the average percentage change from baseline responses. Data are expressed as mean% of the baseline ± SEM. Comparisons were made using a two-way repeated measures analysis of variance followed by post hoc Tukey’s multiple comparisons test. *p < 0.05, ** p < 0.01 and *** p < 0.001 compared to control group. # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to HFD + Aβ group. & p < 0.05, && p < 0.01 and && & p < 0.001 compared to HFD + PBS group.

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oxidative stress and inflammation are the underlying causal mechanisms of AD pathology (Holmes, 2013; Mecocci et al., 1994; Morris and Tangney, 2014). However, whether oxidative damage precedes and directly contributes to the intracellular accumulation of the Aβ1–42 peptide remains a matter of debate (Hernández-Zimbrón and RivasArancibia, 2015). Today, many studies have been conducted to replace chemicals with natural substances, including natural antioxidants from plant sources (Di Pasqua et al., 2010; Ganji et al., 2017b). Herbs contain large amounts of antioxidants, such as secondary metabolites that play an important role in absorbing and neutralizing free radicals (Rangasamy and Namasivayam, 2014). Thy is the one of the major oil constituents in thyme (Aman et al.; Baser, 1992, 1994). Both thyme oil and Thy are beneficial for the antioxidant status of the rat brain (Youdim and Deans, 2000). Thy has demonstrated high ferric-reducing ability and a high reducing power that can be attributed to its higher phenolic contents (Aman et al.). Furthermore, the antioxidant, anti-inflammatory, and anti-lipid peroxidation effects of Thy are protective against toxicity that is related to cellular oxidative stress (Alam et al., 1999; Arab et al.; Kruk et al., 2000). Our experiment demonstrates that Thy possesses antioxidant properties that could improve hippocampal LTP induction in HFD + Aβ rats. Thy prevents HFD-induced obesity in murine models through several mechanisms, including the attenuation of visceral fat accumulation, its lipid-lowering actions, an improvement in insulin and leptin sensitivity, and an enhanced antioxidant potential (Haque et al., 2014). In line with this, the consumption of foods rich in antioxidants, such as certain fruits, legumes, nuts, and vegetables, can counteract obesity (Abete et al., 2011; Kesse-Guyot et al., 2013; Vargas-Robles et al., 2015). In this sense, in our experiment a significant decrease was observed in the level of TG, VLDL and cholesterol in the HFD + Thy + Aβ group. Cholesterol in the brain is involved in a series of interdependent metabolism processes of Aβ including the synthesis, aggregation, neurotoxicity, and elimination. The phosphorylated tau is also considered to be related to cholesterol metabolism (Sun et al., 2015). Accordingly, it has been reported that Aβ would accumulate remarkably in the brain of rabbits fed a high-cholesterol diet (Sparks et al., 1994). In agreement with our results, it has also been demonstrated that blood cholesterol concentrations in rats can be reduced by consuming a polyphenol-rich diet (Boyer and Liu, 2004; Kuo et al., 2015; Osada et al., 2006). In accordance with this, it has been shown that consuming polyphenol rich foods such as Oriental plum may prevent the onset of AD (Lau et al., 2005).

Fig. 9. Thioflavin S staining of amyloid plaques in the hippocampus region (×100).

A maternal HFD can decrease memory formation in mice (Martin et al., 2014; Tozuka et al., 2010; Yu et al., 2010). Accordingly, there is increasing evidence that suggests that HFD in animal models of AD is associated with an increased accumulation of the toxic Aβ peptide and impaired behaviors (Barron et al., 2013). The early phases of obesity are characterized by an increased production of reactive oxygen species and decreased nitric oxide bioavailability (Galili et al., 2007; Vargas-Robles et al., 2015). Obesity-induced blood-brain barrier damage was associated with an upregulation of pro-inflammatory cytokines and increased oxidative stress (Tucsek et al., 2014). The excessive production of oxidants can cause imbalances, termed oxidative stresses (Di Pasqua et al., 2010; Ganji et al., 2017b). The resulting neuroinflammation and oxidative stress in the mouse hippocampus is likely to contribute to the cognitive decline observed in aged obese animals (Tucsek et al., 2014). Increased antioxidant availability may be helpful in preventing or slowing the progress of various oxidative stress-related diseases (Rangasamy and Namasivayam, 2014). Antioxidant and free radical scavenging can protect cell membranes against free radical damage (Burt et al., 2007; Di Pasqua et al., 2007; Di Pasqua et al., 2010). HFD-evoked oxidative stress and mitochondrial damage are mechanisms that can lead to neurodegeneration (Nuzzo et al., 2015). In our study, the rats in the HFD groups exhibited significantly decrease TAC and TTG when compared to those in thymol groups. In our experiment, the MDA and TOS levels in the HFD groups were significantly higher than those in the thymol groups. Oxidative stress markers and antioxidant concentrations were evaluated in several animal studies exploring the role of oxidative stress in neurodegenerative diseases. Oxidative stress is a major risk factor for AD, and has been suggested to be a trigger for AD pathology (Hernández-Zimbrón and Rivas-Arancibia, 2015; Markesbery, 1997). One hypothesis is that

5. Conclusion Our results highlight the potential benefit of Thy as a dietary antioxidant. These results provide preliminary evidence for the effectiveness of Thy in alleviating LTP impairments caused by the increased Aβ levels in AD rats that fed HFD. Antioxidant activity may be the mechanism contributing to the beneficial effects in this model of hippocampal synaptic plasticity. Therefore, natural therapeutic agents based on Thy could be used for the prevention and treatment of oxidative stress-related diseases, such as AD. However, further investigation is necessary to establish its efficacy and potential toxicity in clinical trials.

Conflicts of interest None.

Acknowledgment This study was funded by a grant (Grant Number: 9312186882) of the Hamadan University of Medical Sciences, Hamadan, Iran. 348

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