Twenty four adult male Wistar rats weighing approximately 200 g each were used for this portion of the study. The rats were divided into four groups of six rats each. The first group consisted of untreated controls. The second to fourth groups were injected with kainate. Rats were anaesthetized by ketamine and xylazine cocktail (prepared with 7.5 mL ketamine (75 mg/kg), 5 mL xylazine (10 mg/kg), and 7.5 mL sterile water) and kainate 1 μL of 1 mg/mL was injected through a small craniotomy as previously described (Kim and Ong 2009). The kainate injected rats were killed at 1 day, 1 week and 2 weeks after injection. The animals were deeply anesthetized and decapitated. The lesioned right hippocampi were quickly removed and snap frozen in liquid nitrogen, and kept at −80°C till analyses. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the National University of Singapore.
All reagents for gas chromatography-mass spectrometric (GC-MS) analysis were of analytical grade. Standards for 7-ketocholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, cholesterol 5 beta-6-epoxide, cholesterol 5 alpha-6-epoxide, and 5 alpha-cholestane were purchased from Sigma (St. Louis, MO, USA) and of at least 95% purity. 26 (27)-hydroxycholesterol was obtained from Steraloids (Newport, RI, USA). 5 alpha-cholestane and other compounds were used as internal standards. 7 alpha-hydroxycholesterol-d7, 7 beta-hydroxycholesterol-d7, 26 (27)-OH cholesterol-d5, 7-ketocholesterol-d7, lathosterol, and lathosterol-d4 were purchased from CDN Isotopes (Quebec, Canada). All standards were of high-purity grade (∼95%). Standard solutions of oxysterols, 5-alpha cholestane, and other cholesterol biosynthetic precursors were diluted in ethanol. Formic acid (Lancaster, England), potassium hydroxide, butylated hydroxytoluene, ethanol, acetic acid (Merck, Darmstadt, Germany), and hexane (Tedia, OH, USA) were of analytical grade. Methanol (EM Science, Darmstadt, Germany) and ethyl acetate (Fisher Scientific, Loughborough, UK) were of HPLC grade. N,O-bis(trimethylsilyl)trifluoroacetamide +1% trimethylchlorosilane (BSTFA + TMCS) silylating agent was obtained from Pierce Chemicals (Rockford, IL, USA). Oasis mixed anion-exchange cartridges were from Waters Corp. (Milford, MA, USA).
Extraction of lipids was carried out using the Folch method with slight modification (Folch et al. 1957). Hippocampal specimens of 1 day, 1 week, and 2 weeks post-kainate injected and control hippocampi were homogenized at 4°C with 0.75 mL phosphate-buffered saline (PBS) and 3.25 mL Folch organic solvent mixture (chloroform/methanol 2 : 1, containing 0.05% butylated hydroxytoluene). The homogenates were centrifuged at 1000 g for 10 min at 4°C. The upper phase was discarded and the lower organic phase carefully transferred to a glass vial and evaporated under a stream of N2. 1 mL of 0.5 M KOH (in 100% methanol) and 1 mL of water was added with a mixture of heavy isotopes, 40 ng of 7 alpha-hydroxycholesterol-d7, 40 ng of 7 beta-hydroxycholesterol-d7, 40 ng of 26 (27)-hydroxycholesterol-d5, 80 ng of 7-ketocholesterol-d7, 0.2 μg 5-alpha cholestane, 0.2 μg of lathosterol-d4, 0.2 μg of campesterol-d7, and 0.2 μg of beta-sitosterol-d7 in 25 μL of ethanol were added to the sample and mixed. The tube was purged with argon gas and closed with a Teflon cap. After gentle mixing, samples were incubated at for 15 h at 23°C in order to measure the total (free + esterified) forms of cholesterol, COPs, and oxysterols.
Mixed anion-exchange SPE columns were preconditioned with 2 mL methanol; followed by 2 mL of 20 mM formic acid pH 4.5. 0.5 mL of 0.4 M acetic acid was added to the hydrolysed samples and then neutralized with 0.85 mL of 1 M HCl. The hydrolyzed samples (pH 4.5) were loaded onto the columns, and the columns were washed with 2 mL of 40% methanol/formic acid pH 4.5. After the wash, 2 mL of hexane and then 2 mL of ethyl acetate/hexane (30 : 70) were added to elute cholesterol and oxysterols. The eluted samples were evaporated under a stream of nitrogen. The aliquots were derivatized with 25 μL acetonitrile and 25 μL BSTFA + TMCS for an hour at 22°C. For oxysterols measurement, the derivatized samples were analyzed using Agilent 5975 inert XL mass selective detector. Helium was used as the carrier gas at a flow rate of 0.8 ml/min, derivatized samples (1 μL) were injected splitless into the GC injection port (280°C). Column temperature was increased from 160°C to 300°C at 40°C/min after 1 min at 160°C, then held at 300°C for 6 min. Selective ion monitoring was performed using electron ionization mode at 70 eV (with ion source maintained at 230°C and the quadrupole at 150°C) to monitor one target ion and 2 qualifier ions selected from each compound’s mass spectrum to optimize sensitivity and specificity. Quantification of cholesterol and oxysterols was achieved by relating its peak area of target ion to its corresponding internal standard peak. Statistical analysis of results was done using one-way anova with Bonferroni’s multiple comparison post-hoc test. p < 0.05 was considered significant in all cases.