A monopolar electrode (active) was inserted into the muscle of interest. An identical electrode (reference) was inserted subcutaneously into the lateral and distal-most tendinous portion of the gastroc-soleus complex, ipsilateral

to the muscle studied. A subdermal needle (ground) was inserted subcutaneously into tendinous tissue posterior to and near the reference electrode. Selleckchem RO4929097 Abnormal spontaneous activity in the form of denervation potentials (positive sharp waves and fibrillations) was recorded using an electromyography abnormality score scale (Fig. 5A). F2-isoprostanes and F4-neuroprostanes were measured in ipsilateral brain using the gas chromatography–mass spectrometry method of Morrow and Roberts [25]. Tissue was collected and homogenized in chloroform:methanol containing 0.005% butylatedhydroxytoluene (BHT) to prevent auto-oxidation, dried under a stream of nitrogen, and re-suspended in methanol containing BHT. Esterified F2-isoprostanes in phospholipids were saponified, to free fatty acids from lipids, by adding aqueous potassium hydroxide. Then, the sample was acidified and diluted

with water. Next, deuterated-F2-isoprostane internal standard was added to the mixture. For the measurement of free F2-isoprotanes/F2-isofuranes in plasma, the extraction and hydrolysis steps were omitted, and the sample was simply acidified, diluted, and the internal standard added. The mixture was subsequently run Etomidate on

a silica column to separate isoprostanes/isofuranes find more from bulk fatty acids. The eluate was converted to pentafluorobenzyl esters, by treatment with pentafluorobenzyl bromide to improve separation efficiency. The mixture was then subjected to thin layer chromatography to remove the excess pentafluorobenzyl bromide and unreacted fatty acids. The F2-isoprotane/isofurane fraction was extracted using ethyl acetate, and analyzed. F2-isoprostanes were quantified by peak height, the data were corrected with the internal standard, and results were calculated as nanogram of F2-isoprostanes per mL of plasma or per gram tissue. F4-neuroprostanes, a lipid peroxidation product of docosahexaenoic acid was also determined some modifications of the F2-isoprostane method. Briefly, 100–200 mg tissue was homogenized in ice-cold Folch solution containing BHT. Lipids were then extracted and chemically hydrolyzed with 15% KOH. After acidification with HCl and addition of a stable isotope-labeled internal standard, 8-iso-prostaglandin F2α-d4, F4-neuroprostanes were applied to a C18 Sep-Pak cartridge and a silica Sep-Pak column for further purification. Unlike the F2-isoprostane assay, the washing step for silica columns used an ethyl acetate/heptane (75:25) mixture instead of pure ethyl acetate because of the polarity difference between F2-isoprostanes and F4-neuroprostanes.