[1] 21w The Effect of Picein on Inhibitory Avoidance Memory and Activity of Antioxidant Enzymes in Hippocampus of Male Rats with Scopolamine-Induced Injury

[1] 263w Alzheimer disease (AD) is a common neurologic disorder, impairing memory and spatial perception. Consistent with the extensive search for its treatment, we investigated the effect of Picein on inhibitory avoidance memory, lipid peroxidation, and the activity of hippocampal antioxidant enzymes in rats. Forty adult male Wistar rats were randomized into control group (no intervention), model group (intraperitoneal injection of 3-mg/kg scopolamine), and three interventional groups (1.5-, 2.5-, and 5-mg/kg intraventricular Picein, once a day for 7 days, 24 h after scopolamine injection). After behavioral test, the rats' hippocampus was isolated for measuring oxidative stress markers, including enzymes superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPX), catalase (CAT), and total antioxidant capacity (TAC). One-way ANOVA was used for comparing numeric variables among the groups using SPSS v.21. The results showed scopolamine decreased SOD, GPX, and CAT enzymes, and TAC level, and increased MDA level, compared with the control group (P < 0.001) that confirmed the scopolamine-induced AD model. The two doses of 2.5-and 5-mg/kg Picein increased latency for entering the dark room, compared to the scopolamine group (P < 0.05), making them similar to the control group. The number of entries into the dark room in the 2.5-mg/kg Picein reduced and approached the control group (P < 0.05). The 2.5-mg/kg Picein decreased MDA and increased SOD, GPX, and TAC, more than 5 mg/kg Picein, both different than scopolamine; only 2.5-mg/kg Picein had different CAT, compared to scopolamine group (P < 0.05). In conclusion, by lowering oxidative stress in the hippocampus, Picein was able to prevent the scopolamine-induced impaired learning and avoidance memory in rats.

[1] 115w Alzheimer disease (AD) is one of the most common neurodegenerative disorders (NDDs), the top cause of dementia in the elderly, and the sixth leading cause of death in the USA, fifth among age ≥ 65 years [1]. It is estimated that the current rate of 6 million Americans with AD will reach to about 14 million by 2050; this increased prevalence will be associated with high disease burden and medical costs [1,2]. Furthermore, the significant dementia, memory loss, and cognitive dysfunction impair the quality of life of patients and their family members/caregivers [3]. Most of the current therapeutic strategies are only symptom-reliever, and research is continued on disease-modifying therapies, which tackle the disease pathogenesis [4].

[2] 198w Oxidative stress, induced by the imbalance between antioxidants and reactive oxygen species (ROS), is one of the important pathogenic factors of AD, which leads to mitochondrial dysfunction, altered permeability of cellular membrane and calcium hemostasis system, apoptosis, and increased production of metabolic enzymes [5]. Accordingly, intracellular antioxidant enzymes, like superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) are the first-line antioxidants, which eliminate free radicals from the body [6]; measuring these enzymes can determine the involvement level of oxidative stress in the synaptic loss, resulting in AD [7]. Total antioxidant capacity (TAC) is another marker of oxidative stress, produced by DNA oxidation; its decrease is suggested as a helpful tool to discriminate AD from healthy controls [8]. Additionally, the brain's phospholipids and unsaturated free fatty acids, known as lipid peroxidation (LPO), can be destroyed by the oxidative stress and its level can be measured by malondialdehyde (MDA), a byproduct of hydroperoxide decomposition [9]. Neuronal damage, induced by AD, can be microscopically observed in the hippocampus, cerebral cortex, and basal ganglia. Considering the critical role of hippocampus in memory and learning, it is usually studied for investigating the effect of therapeutic agents in animal models of AD [10].

[3] 136w For inducing cognitive impairment, mimicking AD in rats, previous studies have used the intraperitoneal (IP) injection of scopolamine hydrobromide and showed that it can impair the rat's memory (measured by behavioral tasks with the Morris Water Maze test) and increase the level of oxidative stress markers (involved in the pathogenesis of AD), 24 h after administration [11]; we have also shown that it can result in hippocampus shrinkage in rats, as well [12,13]. Neuroscience research suggests that this muscarinic cholinergic receptor antagonist neurotransmitter permeates the blood-brain barrier, causes cholinergic dysfunction, and increases amyloid-beta (β) deposition, the main hallmarks of AD [14], which lead to production of free radicals, membrane damage, reduced cell viability, and ultimately cell death [15]. Therefore, the IP injection of scopolamine is known as a successful model of AD induction method in rats.

[4] 272w Considering the role of oxidative stress on the structural and functional impairments in AD, exogenous antioxidants, including nutritional and herbal compounds, have been suggested as supplementary therapies for symptom relief and/ or progression halt in NDDs; the mechanism of action for these compounds includes neutralizing free radicals and reducing the plaque formation by influencing sequestering metal ions [10,16,17]. In a cellular model of neuroblastoma, scientists found neuroprotective effects of the phytochemical compound Picein (PubChem ID: 92123), a phenolic (nonsalicylic) glycoside, with the formula C14H18O7 [13,18]. Review of evidence showed the potential neuroprotective effect of Picein with the need to confirm this hypothesis in an animal model, not investigated to date [19]. Considering the potential neuroprotective role of Picein and lack of studies in this regard, the present study aimed to investigate the effect of Picein in scopolamine-induced AD rats; three main outcomes measured included: (1) inhibitory avoidance memory, measured by passive avoidance test; (2) the activity of hippocampal antioxidant enzymes, including SOD, GPX, and CAT, TAC, as well as LPO measurement by MDA, and (3) neuronal density of hippocampus, evaluated by microscopic evaluation of the brain sections. For accurate comparisons, we selected two control groups, one control group with no intervention (normal rats) and another control group with model effect to investigate the effect of scopolamine (mimicking AD) and compare the results of intervention (Picein) with both control groups. In this way, the results of this study can help understand both the effect of scopolamine on AD parameters in rats and different doses of Picein on scopolamine-induced AD rats, compared to AD model (model group) and normal rats (without AD; control group).

[1] 48w Evaluating drug toxicity showed that doses of 50 and 100 mg did not cause death or any change in autonomic symptoms/ behaviors. Over 10 days, a 200 dose caused 50% mortality and was selected as LD 50 dose. The 400 dose caused a mortality of greater than 50%.

[2] 103w Figure 2A shows the latency time for entering the dark room (black color) for each of the control, Picein, and model groups. In the control group, the latency time was shorter than the Picein groups, which indicated normal memory and low anxiety levels. In model group, the latency time reduced, compared to the control and Picein groups, reflecting the negative effects of scopolamine on memory and increasing anxiety. Picein groups 2.5 mg/kg and 5 mg/kg had the highest latency duration, compared to scopolamine group, suggesting the positive effect of Picein on memory and reducing anxiety (F[2,56] = 75.45, P < 0.001; Fig. 2).

[3] 96w Figure 2B shows the number of entries into the dark chamber in different experimental groups, which was very few in the control group, indicating healthy memory and the ability to avoid entering the undesirable environment (dark chamber). The scopolamine group exhibited the highest number of entries into the dark chamber, demonstrating the negative effect of scopolamine on learning and memory. In the groups treated with Picein, at a dose of 2.5 mg/kg, the number of entries into the dark chamber is further reduced, approaching the levels of the control group (F[1,96] = 65.35, P < 0.001).

[4] 50w The concentration of MDA increased in scopolamine and 1.25-mg Picein groups, compared to the control group (p ≤ 0.001) and decreased in 2.5-and 5-mg/kg Picein groups, compared to the scopolamine group (p > 0.05) with the greatest reduction at the dose of 2.5 mg/kg (mean difference of 5.55 nmol/mg protein).

[5] 84w Activity of the SOD, GPX, and CAT enzymes, as well as TAC significantly reduced in scopolamine and 1.25-mg Picein groups, compared to the control group (p ≤ 0.001). In addition, 2.5-and 5-mg/kg Picein significantly increased the activity of SOD, GPX, and TAC, compared to scopolamine group (p < 0.05) with the highest effect observed in 2.5-mg/ kg group; considering CAT, only 2.5-mg Picein was different from the scopolamine group. Figure 3 shows a better illustration of the difference in the values of these enzymes.

[6] 115w Microscopic evaluation, as illustrated in Fig. 4, showed that the mean number of hippocampal neurons, in four hippocampus regions, reduced in scopolamine group, compared to the control group (P < 0.001) and increased in Picein groups, compared with the saline-scopolamine group (F[4,138] = 34.078, P < 0.001, Fig. 4). In all regions, the three Picein groups were different from each other with the highest neuronal density observed in 2.5 mg dose, followed by 5 mg, and then in 1.25 mg group (Fig. 4). The cresyl violet staining of the microscopic images, taken from the sagittal section of all four hippocampus regions, including CA1, CA2, CA3, and DG are also shown in this figure (Fig. 4).

[1] 318w In this study, we examined how scopolamine with and without Picein influenced memory avoidance, the activity of antioxidant enzymes, and neuronal concentration in the Fig. 2 The effects of treatment by Picein on passive avoidance latency (A) and number of entries to the dark room (B), in scopolamine-exposed rats in the five study groups, including control, scopolamine (sco), and scopolamine plus three doses of Picein (1.25, 2.5, and 5 mg/kg) groups. For intergroup comparisons: **P < 0.05, ***P ≤ 0.001 versus control group, + + P < 0.01, and + + + P < 0.001 versus scopolamine hippocampal region of rats, following scopolamine-induced injury in male rats. Comparing the results in scopolamine group (which received scopolamine and saline) with the control group (which did not receive any intervention) showed that scopolamine was able to decrease the activity of CAT, GPX, and SOD enzymes and increase MDA concentrations, as well as LPO of hippocampal neurons. These results confirm the oxidative stress, induced by scopolamine. Budzynska and colleagues also suggested that scopolamine injection results in decreased activity of anti-oxidant enzymes (SOD and GPX) and increased concentration of MDA (which reflect the LPO level) [26]; these results are in line with the results of the present study, indicating the induction of oxidative stress and LPO by scopolamine in rats, which confirm that IP injection of scopolamine is an appropriate animal model for AD. Furthermore, the results of the behavioral test in our study showed reduced latency duration vs. control group, which suggest impaired passive avoidance memory by administering scopolamine; these results are in line with the results of the previous studies and indicate scopolamine-induced memory deficit, mimicking AD [26]. Also, in our previous experiments, we showed scopolamine as an appropriate animal model of AD, in rats [10,20,21], which in line with the results of the present study and previous studies, show scopolamine as a successful model for AD induction in rats.

[2] 349w We used two control groups in this study, one without any interventions (control group, mimicking normal rat with no Fig. 3 Comparing the activity of antioxidant enzymes and the malondialdehyde level following induction of brain injury with scopolamine and treatment with different doses of Picein (1.25, 2.5, and 5 mg/kg/day). For intergroup comparisons: + P < 0.05, + + P < 0.01, and + + + P < 0.001 versus scopolamine-treated group; *P < 0.05, **P < 0.01, and ***P ≤ 0.001 versus control disease) and the other receiving scopolamine + saline (model group; mimicking AD); in order to observe the effect of treatment with three doses of Picein (which received scopolamine + 1.25-, 2.5-, or 5-mg/kg Picein), compared with both normal conditions (control) and acute AD (model). The results showed that 2.5 and 5 mg/kg reduced the MDA concentration and increased the antioxidant enzymes (GPX, SOD, and TAC), compared to scopolamine group, and were not different from the control group, suggesting the great effectiveness of Picein as much as reaching normal levels (without disease). But CAT enzyme level was only different in 2.5-mg/kg Picein group, compared to scopolamine group, which may refer to the dose-dependent effect of Picein. These results confirmed that Picein could reduce the oxidative stress and LPO, induced by scopolamine in rats, which suggests Picein as an effective anti-oxidant agent. The reduced oxidative stress has been used in rat models of AD treatment in previous studies, as well [17,27], the results of which are in line with the results of the present study. Also, histologic evaluation in the present study showed that Picein increased the neuronal density, which had been reduced by scopolamine injection; interestingly, Picein made the neuronal density similar to normal (control group). This finding confirms the neuroprotective effect of Picein in a cellular model. Also, the behavioral test showed that the two higher doses of Picein reduced the effect of impaired passive avoidance memory, induced by administering scopolamine and approached it to normal levels (control group); these results confirm the favorable effect of Picein on scopolamineinduced memory impairment in an animal model.

[3] 79w Picein is one of the compounds with scant evidence available in the literature on its effects on cellular and show live neurons in the hippocampus. Scale bar is 20 μm in all panels. Below shows the mean values in the groups: (1) control; (2) saline + Sco 3 mg/kg, IP; (3) Picein 1.25 mg/kg + Sco; (4) Picein 2.5 mg/kg + Sco, and (5) Picein 5 mg/kg + Sco. *P < 0.05, **P < 0.01, and ***P ≤ 0.001

[4] 410w animal models [19]. Few cellular models have shown its antioxidant effects [19,28]. In herbal studies, Picein, the precursor of piceol, has been suggested as the indicator of plant stress in Norway spruces (Picea abies) [29], associated with a stronger defense against fungal pathogens [30] and bacteria [31] in the tree. The presence of Picein in another similar tree, white spruce (Picea glauca), was found effective on a forest insect pest [32]. These studies suggested the anti-oxidative and antibacterial effects of Picein. However, as these results were obtained on plants (trees), we cannot generalize the results to animals or humans. In addition, Salix species, one of the herbs containing Picein, which belongs to Salicaceae family, has significant analgesic, antiinflammatory, antioxidative, anticancer, cytotoxic, antidiabetic, antimicrobial, antiobesity, antimigraine, hepatoprotective, and neuroprotective activities [33][34][35][36]. Salicylic acid, aspirin precursor, is also extracted from willow bark and leaves by animals and ancient humans for its analgesic, antipyretic, and antiinflammatory properties [37]. However, the anti-oxidative activity of Salix sp. has been attributed to salicin [38] and among different species of Salix, found rich in salicylic glycosides (salicin, salicortin and tremulacin, flavanones, maringenin, chalcone isosalipurposide, and catechin), Picein, a phenolic (non-salicylic) glycoside, was only identified scarcely (only in 10% of these species) [39]. The few studies on the anti-oxidative effects of the compounds extracted from Salix sp. also failed to indicate any favorable effect for Picein, which is contrary to the results of the present study; although the extraction method and studied cells differed among these studies. In the study by Jeon and colleagues, no cytotoxic effect was established for Picein against brine shrimp and a human lung cancer cell line (H1299) [40]. Also, Picein was found unable to inhibit cell proliferation in an immortalized human nontumorigenic keratinocytes (HaCaT) [36]. Another study also showed no anti-oxidative activity for Picein, extracted from Picrorhiza kurroa [28]. As far as we are concerned, no study, other than that by Kesari et al. [13] and our study, has studied the neuroprotective effect to the Picein compound of Salix or any other plant. In the study by Kesari and colleagues, the researchers showed that treatment of neuroblastoma SH-SY5Y cells with Picein decreased the menadione-induced ROS levels and recovered the mitochondrial activity to normal levels that indicated the potential of Picein to be used as a neuroprotective agent [13]. Therefore, evidence on the potential neuroprotective property of this compound is missing from the literature, and we cannot compare our results with a similar study.

[5] 143w Considering the association of the pathophysiology of AD with oxidative stress, a number of studies have evaluated the effect of different substances on oxidative stress; many have shown affirmative results. One of the strengths of the present study was evaluation of antioxidative and neuroprotective effects of this compound, in addition to the rat's behavior, which provides a stronger evidence (both cellular and animal models) for suggestion of Picein as a novel treatment of AD. Furthermore, we evaluated the toxicity level of Picein in this study, as well, for identifying the most appropriate dose. Also, two groups were used for comparison, control and model, in order to observe whether the efficacy of Picein is as much as reaching normal levels or not. Nevertheless, this study had some limitations, most of which were related to the scant evidence available in the literature on this compound.

[1] 99w According to the results of the current research, 7 days of treatment with Picein (1.25, 2.5, and 5 mg/kg/day) appears to be capable of enhancing antioxidant defense, lowering LPO levels, controlling cell death, and improving passive avoidance memory deficit, caused by scopolamine in rats. Memory and cognition are both significantly impacted by the hippocampus and the increased neurogenesis, induced by Picein suggest this agent capable of protecting against the neuronal and behavioral deficits that scopolamine had caused in the relevant animal model. Accordingly, it is suggested to consider Picein in future research on the novel treatment options for NDDs.

[1] 21w The protocol of this research was confirmed by the Ethics Committee of Neuroscience Research Center, Golestan University of Medical Sciences (IR.GOUMS.REC.1400.161).

[2] 50w The drugs and chemicals, used in this study, included scopolamine hydrobromide (Tocris, Great Britain) and Picein (C14H18O7) (PubChem ID: 92123) USA. Catalase (CAT) activity assay kit (ZX-44102-96), superoxide dismutases ELISA kit (SOD) (RK03959), MDA/malondialdehy-deELISA kit (RK09070), glutathione peroxidase 1 (GPX1) (ENZ-186) ZellBio kit (ZellBio, Germany), Cresyl Violet (10510-54-0) Sigma-Aldrich, USA.

[3] 112w For this experiment, 40 adult (12-week-old) male Wistar laboratory rats that weighed between 120 and 180 g each, were acquired from the Pasteur Institute in Amol, Iran, and housed in transparent plastic cages at the animal house of Golestan University of Medical Sciences. The sample size was determined at 40, similar to previous projects [10,20]. Eight rats were housed in each cage. Except for when they were being experimented on, they always had free access to food and drink. The animals were housed in light-dark cycles of 12 h (from seven in the morning to seven in the evening), at room temperature (22 ± 3 °C), with no exposure to noise pollution.

[4] 237w After adaptation to the new laboratory conditions, eight rats were selected for each group and randomly categorized into five groups: Control group: No medication and no surgery, were trained with shuttle box on day 1, underwent memory test on day 15, and got their brains extracted on day 16. Model group: were trained with shuttle box on day 1, underwent stereotaxic surgery on day 2, after a 7-day recovery from stereotaxic surgery, the rats received an intraperitoneal (IP) injection of 3-mg/kg scopolamine to induce memory impairment; on the next day, 5-µl normal saline was injected into the right lateral brain ventricles, once a day for 7 days. One day after the end of injections, behavioral test was performed, resulting in a total of 16 days (Fig. 1). Intervention groups: First they received scopolamine, in the same manner as the model group. Twenty-four hours later, Picein, dissolved in normal saline, was injected into the right lateral brain ventricles (using 10-µL Hamilton syringe and needle number 21); the doses of Picein were 1.25, 2.5, and 5 mg/kg in groups 3, 4, and 5, respectively. The injections were given at 9 a.m., once a day for 7 days. Picein doses were selected based on acute toxicity and lethal dose (LD) of Picein in this study. In all experiments, the experimenter was blind to the group allocations. Figure 1 provides a better illustration of the groups and timelines in each group.

[5] 46w Fig. 1 The study groups and interventions performed at each day Note. Based on the results of our previous experiments, we found no difference in the results obtained from control and sham groups [10]; therefore, we omitted sham from this study and only considered control group.

[6] 85w The stereotaxic surgery was performed in the same way as our previous study [20] using stereotaxic device with the indicator set at Bergma. The same landmarks were marked and drilled (DV = -4/2, AP = -0/8; according to the Paxinos and Watson atlas), the cannula was inserted into the skull and fastened with dental acrylic. After dental acrylic was dried and hardened, the animals were removed from the stereotaxic device, transferred to a warm environment, and kept in separate cages until they gained full consciousness.

[7] 189w The passive avoidance test was performed in the same way as in our previous studies [20,21]; in the first phase (acclimation), the rats were introduced to the passive avoidance apparatus over two consecutive days, prior to the training phase. During this period, no shocks were administered, allowing the animals to acclimate to the environment. The next phase (training phase) took place on day 3, when each rat entered the light chamber first and received a mild foot shock (50 Hz, 1 mA, 3 s) upon entry into the dark chamber. The rat was then returned to its home cage, and the process was repeated. Rats that refrained from entering the dark chamber for at least 120 s after the shock were considered to have successfully learned the passive avoidance task. The final phase (testing) was conducted on day 16, when no shock was administered and the latency period was recorded (maximum of 300 s). As avoiding entering the dark room cannot differentiate between the anxiety/stress (of the irritant) and memory, we also calculated the number of entries to the dark room (during the 300 s) for examining the anxiety/stress.

[8] 94w After completion of the behavioral tests, at the end of the 4th week, the animals in each group were anesthetized with LD of chloroform, and their heads were removed with a rodent guillotine; the brain was completely removed from the skull and placed on ice quickly. Then, the hippocampus was carefully separated from the rest under a stereoscope (Olympus, Japan). The tissue was homogenized based on the method explained previously [22]; the supernatant was removed with a sampler and investigated for the hippocampal values of oxidative stress factors (MDA, SOD, GPX, CAT, and TAC).

[9] 323w A. Measurement of the hippocampal MDA: The extracted hippocampus was immediately weighed and homogenized with 10 mL of 1.5 KCL solution per 1 g of tissue; 0.5 mL of the homogenized solution was removed, 2.5 mL of 3% MDA was added, and kept at 37 °C for 10 min. It was then centrifuged at 3000 rpm for 10 min; 0.5 mL of the supernatant was removed after centrifugation, 3 mL of 0.1 phosphoric acid solution plus 1 mL of 0.67% TBA solution were added to each, and they were placed in boiling water for 45 min. The tubes were cooled in ice and 4 mL of butanol was added to each. After vortexing, they were centrifuged at 2000 rpm for 20 min. Finally, the MDA concentration (nmol/g/wet tissue) was evaluated based on reaction with thiobarbituric acid (TBA; Merck, Germany), after adsorption with a wavelength of 532 nm, contracting the numbers obtained by spectrophotometry, and absorbing the linear equation of the standard curve. Absorption was measured by spectroscopy and compared with the standard curve. According to the enzyme-linked immunosorbent assay (ELISA) kit protocol, the initial amount of tissue was considered the same in all samples [23]. B. Measuring the activity of SOD enzyme in hippocampal tissue: The pyrogallol autoxidation method was used to measure the activity of the SOD enzyme. For this purpose, 50 mM tris-hydrochloride buffer (with pH = 8.2), 1 mM tris-hydrochloride buffer (EDTA), and 0.2 mM pyrogallol were used. EDTA (2900 μL) was mixed with 50 μL of the supernatant; then, 50 μL of pyrogallol solution was added to the above solution, and the adsorption changes were read for 5 min at 30-s intervals at a wavelength of 420 nm. To measure pyrogallol autoxidation, 50 μL of distilled water was added to the supernatant alone as a control; the absorption was then read at 420 nm, and the percentage of inhibition of pyrogallol autoxidation was calculated by the following Eq. [23]:

[10] 342w Percentage of inhibition of pyrogallol autoxidation = dA∕dt_blank -dA∕dt_sample)∕dA∕dt_blank × 100 C. Measuring the activity of GPX: This experiment was performed using the working method, defined in the Ransel kit, produced by Randox Company [24]. For this purpose, first, the hippocampal tissue was homogenized in a cold buffer (e.g., phosphate buffer) and centrifuged to obtain the supernatant, containing the enzyme. The assay was conducted using a reaction mixture, containing reduced glutathione (GSH), glutathione reductase (GR), and NADPH; the activity of GPX enzyme was determined by monitoring the decrease in absorbance of NADPH at 340 nm, as it converted to NADP⁺, using ZellBio kit (ZellBio, Germany), and expressed as units per milligram of protein (U/mg protein). D. Measuring the activity of catalase enzyme in hippocampal tissue: The CAT activity was measured (by blue method) using hydrogen peroxide substrate. The reaction was performed in a 3-mL coat. The reaction mixture consisted of 980 μL of a 30 mM solution of hydrogen peroxide, 2 mL of a 50 mM sodium phosphate buffer (with pH = 7), and 20 μL of the enzyme sample (supernatant). The reaction was started by adding substrate and the adsorption changes were measured for 2 min at 240 nm. CAT enzyme activity was calculated using the extinction coefficient (M-1CM-1 4/39), according to the Beer-Lambert law (A = cdc), and reported as U/g tissue. By definition, a unit of CAT is the amount of an enzyme that decomposes one micromole (H 2 O 2 ) in 1 min at 25 °C [25]. E. Measuring the activity of TAC enzyme in hippocampal tissue: For measuring the hippocampal values of TAC, the hippocampal tissue was immediately homogenized in ice-cold phosphate-buffered saline (PBS). After homogenization at a ratio of 1:10 (w/v) (e.g., 100 mg tissue in 1 mL buffer), the homogenate was centrifuged at 10,000-12,000 × g for 15 min at 4 °C to remove cellular debris; the resulting supernatant was carefully collected and used for TAC analysis. For long-term storage (to prevent enzyme degradation), the supernatant was stored on ice or at -80 °C.

[11] 131w Sectioning was performed using a rotary microtome (Leitz, Germany), which cut tiny sections of brain tissue by its sharp blade. For this purpose, we put the block in the microtome device and fixed it with screws to prevent the block moves during cutting. Then, we turned on the device and selected the desired thickness; for acquiring 6-μm-thick sagittal sections, we placed the cannula tip in the lateral ventricle; the appropriateness of its placement was tested microscopically, in the same way as in our previous study [20]. The serial sections provided a total of 25 cuts from the hippocampus of each rat (each block), all of which were used for assessment of neurons in the hippocampus tissue. After cutting the blocks and collecting the slices, 5 lams were selected from each block.

[12] 86w The method of histological processing has been explained before [20]. After staining the brain slices with cresyl violet (0.02%, Sigma, USA), images were taken from hippocampal neurons, localized in the CA1, CA2, and CA3 areas, and dentate gyrus (DG) (using an Olympus BX53 light microscope, Japan and an Olympus DP73 digital camera, Japan at a 400 × magnification). The proper grids were overlaid on the images using ImageJ software; an area of 30,000 μm 2 was selected for calculations, and numbers of neurons were counted manually.

[13] 108w The collected data were input into SPSS version 21.0 (IBM Corp. 2012. Armonk, NY: IBM Corp), used for the statistical analysis. For numeric variables, first the normal distribution of variables was evaluated using Kolmogorov-Smirnov test. Homogeneity of variances was also tested using test of homogeneity of variances. As the results of this test showed a P value > 0.05, mean and standard deviation (SD) were used and for comparison among the groups, parametric test of one-way analysis of variance (ANOVA) was used; post hoc analysis and the Newman-Keuls test were used, when ANOVA results were significant. In these tests, the p value < 0.05 was considered statistically significant.

[1] 66w In a separate experiment, 12 male rats were used to assess the drug toxicity. The rats were divided into four groups of three. They received intraventricular injection of Picein, ten times the study dose of 5 mg, doubled in each experiment (50, 100, 200, and 400 mg) for 7 days. The animals were monitored for mortality, alertness, muscle tone, reflexes, and autonomic symptoms during this time.