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On day 10 after tibial crush treated with saline, nicotine, or nicotine plus MLA, rats ( n = 18) were anesthetized with sodium pentobarbital (50 mg/kg) with additional intravenous boluses of pentobarbital as needed and put on a warm blanket to keep the body temperature close to 37C

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These pieces were incubated in Ca/Mg-free Hanks balanced salt solution (HBSS, SigmaAldrich) containing 30 mM ethylenediaminetetraacetic acid (EDTA, SigmaAldrich) and 1.5 mM dithiothreitol (DTT, SigmaAldrich) at 37 C with shaking for three cycles of 10 min each

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