Phosphoric acid

The cells were treated with varying SME concentrations (12.5, 25, 50, 100, and 200 µg/mL) for 2 h, followed by stimulation with LPS (1 µg/mL) for 24 h. Following incubation, the cell culture supernatants were gathered and combined with Griess reagent, composed of 1% sulfanilamide (#S9251), 0.1% N-1-naphthylenediamine dihydrochloride (#N9125) (Sigma-Aldrich), and 2.5% phosphoric acid (#1.00573.1000, Merck Millipore, Burlington, MA, USA), and then incubated at RT for 5 min. The absorbance of each sample was read at 540 nm using a Synergy HTX microplate reader (BioTek Instruments). NO production was quantified by comparing the absorbance values to a standard curve generated using a nitrite standard solution.

ChemicalsSafranal and quinolinic acid (QA) were purchased from Fluka (St. Gallen, Switzerland) and Sigma (St. Louis, US), respectively. DTNB (2,2'-dinitro-5, 5'-dithiodibenzoic acid), tripyridyltriazine (TPTZ), TBA (2-thiobarbituric acid), Tris (hydroxymethyl) aminomethane (Trizma base), ethylene diamine tetraacetic acid disodium salt (Na₂EDTA), t-octylphenoxypoly-ethoxyethanol (Triton X-100), sodium lauroyl sarcosinate (sarkosyl), ethidium bromide, methanol, sodium acetate, glacial acetic acid, phosphoric acid, potassium chloride, ferric chloride, ferrous sulfate, chloral hydrate, and hydrochloric acid were obtained from Merck (Dramstadt, Germany). Low melting point (LMP) and normal melting point (NMP) agarose were purchased from Biogen (Mashhad, Iran) and Fermentase (Glen Burnie, US), respectively.
AnimalsAdult male Wistar rats weighting 250-300 g from the Central Animal House of Mashhad University of Medical Sciences (Mashhad, Iran), were used throughout the study. The animals were housed in the same room under a constant temperature (22±2 °C) and standard conditions of a 12h light/dark cycle with free access to food pellets and tap water, available ad libitum. The experimental protocol was approved by the Animal Care and Use Committee (87534), Mashhad University of Medical Sciences and was performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Treatment scheduleThe animals were randomly divided into five different experimental groups of seven animals each. Group 1 (sham group) received single intraperitoneal (IP) injection of normal saline (10 ml/kg) plus 1 µl of normal saline which was infused into the left hippocampus, 30 min later. Group 2 (QA group) received single IP injection of normal saline (10 ml/kg) plus intrahippocampal (IH) administration of QA (300 nmol/1 μl/rat), 30 min later. Groups 3-5 (treatment groups) were injected by safranal (72.75, 145.5, and 291 mg/kg, IP), 30 min prior to QA administration (300 nmol/1 μl/rat, IH).
Intrahippocampal administration of QAThe animals were anesthetized with chloral hydrate (400 mg/kg, IP and then positioned in a stereotaxic apparatus (Stoelting, US). After exposing the bregma suture, a small burr hole was made through the skull to permit access of microinjection needle into the left ventral hippocampus according to the brain atlas of Paxinos and Watson (AP 3.7 mm, ML 2.4 mm, and DV 3.2 mm) (27 ). Using a 29-gauge stainless steel needle connected to a Hamilton syringe (Bonaduz, GR, Switzerland), one microliter saline solution containing 300 nmol QA (or vehicle alone as control) was unilaterally microinjected into the left ventral hippocampus region over a period of 1 min and left in situ for another 1 min to prevent back diffusion of the injected drug solution (28 (link), Figure 1). Following surgery, the animals were kept warm to recover from surgery and maintained in suitable situation for 24 hr. After that, the animals were decapitated, brains were quickly removed, kept in ice-cold saline, and the extracted hippocampi were immediately frozen in liquid nitrogen and maintained at -80°C until processing. The injection site was also verified using 1 µl methylene blue and anatomical observation.
The left hippocampus portion was gently homogenized in ice-cold phosphate buffered saline (0.1 M, pH 7.4) to give a 10% homogeny suspension and used for biochemical and comet assay.
Ferric reducing/antioxidant power (FRAP) assayThe basis of FRAP assay is reducing the colorless Fe^III-TPTZ complex to blue colored Fe^II-TPTZ complex, by action of electron donating antioxidants in biological samples (29 (link)). The FRAP reagent consists of 300 mM acetate buffer (pH=3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃.6H₂O in the ratio of 10:1:1.
Briefly, 50 μl of homogenate was added to 1.5 ml freshly prepared and prewarmed (37ºC) FRAP reagent in a test tube and incubated at 37ºC for 10 min. The absorbance of the blue colored complex was read against reagent blank (1.5 ml FRAP reagent + 50 μl distilled water) at 593 nm. Standard solutions of Fe^II in the range of 100 to 1000 mM were prepared from ferrous sulphate (FeSO₄.7H₂O) in distilled water. FRAP values were expressed as nmol ferric ions reduced to ferrous form/mg tissue (29 (link)).
Total sulfhydryl (SH) groups measurementTotal thiol content was estimated based on the Ellman method (30 (link)). In this method, SH groups react with chromogenic DTNB and produce a yellow-colored dianion (5-thio-2- nitrobenzoic acid, TNB), which has peak absorbance at 412 nm.
Briefly, 1 ml Tris-EDTA buffer (0.1 M Tris, 10 mM EDTA, pH=8.6) was added to 50 µl homogenate sample in 2 ml cuvettes. Sample absorbance was read at 412 nm against Tris-EDTA buffer alone (A₁), then 20 µl DTNB reagent (10 mM in methanol) was added to the mixture. Following 15 min incubation at room temperature, the sample absorbance was read again (A₂). DTNB reagent absorbance was also read as a blank (B). Total thiol concentration was calculated by the following equation and expressed as nmol/mg tissue (22 (link)).
Total thiol concentration (mM) = (A₂-A₁-B) × (1.07/0.05) × 13.6
Thiobarbituric acid reactive species measurementHippocampal lipid peroxides formation was measured as malondialdehyde (MDA), which is the end product of lipid peroxidation and reacts with thiobarbituric acid (TBA) as a TBA reactive substance (TBARS) to produce a pink colored complex which has peak absorbance at 535 nm (31 ). In brief, 1 ml of homogenate sample was mixed with 2 ml of TCA-TBA-HCl reagent (15% TCA, 0.67% TBA, and 0.25N HCl) and heated for 45 min in a boiling water bath. After cooling, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was collected, and the absorbance was read against blank, at 535 nm. The amount of MDA produced was calculated, using a molar absorption coefficient of 1.56×10⁵ M^-1cm^-1 and expressed as nmol/g tissue (32 (link)).
Alkaline single cell gel electrophoresis (SCGE) assayThe in vivo alkaline SCGE (comet) assay was conducted based on the method described by Sasaki et al with some modifications (33 (link)). In brief, 10 µl of the hippocampus cells suspension, prepared as above, was mixed with 90 µl LMP agarose (0.5% in physiological saline), and the mixture was quickly layered over a microscope slide precoated with a layer of 100 µl NMP agarose (1% in physiological saline), the slides were then covered with a cover slip, and placed on ice to allow agarose to gel. Finally, another layer of LMP agarose was added on top. The slides were immersed immediately in a chilled lysing solution (pH 10) made up of 2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, and kept at 0^○C in the dark overnight. Then, the slides were placed on a horizontal gel electrophoresis platform and covered with a chilled alkaline solution made up of 300 mM NaOH and 1 mM Na₂EDTA (pH>13). They were left in the solution in the dark at 0^○C for 40 min, and then electrophoresed at 0^○C in the dark for 30 min at 25 V and approximately 300 mA. The slides were rinsed gently three times with 400 mM Trizma solution (pH 7.5) to neutralize the excess alkali, stained with 50 µl of 20 mg/mL ethidium bromide, and covered with a cover slip.
One hundred nuclei per organ from each animal (50 nuclei on one slide) were examined and photographed using a fluorescence microscope (Nikon, Kyoto, Japan) at 400X magnification equipped with an excitation filter of 520-550 nm and a barrier filter of 580 nm. Undamaged cells resemble an intact nucleus without a tail, and damaged cells have the appearance of a comet. The percent of DNA in the comet tail (%tail DNA), which is an estimate of DNA damage, was measured using a computerized image analysis software (CASP software).
Statistical analysisThe statistical analysis was performed using Prism 5.00 for Windows software (Graph-Pad Software, San Diego, CA). Data were expressed as mean±SEM. Comparisons between the study groups were made using one-way ANOVA followed by Tukey-Kramer post-hoc test for multiple comparisons. The p-values less than 0.05 were considered to be statistically significant.