One week prior to the initiation of CIH, all rats were instrumented with an osmotic minipump (Alzet Mini-Osmotic Pump Model 2002, Durect Corporation, Cupertino, CA) implanted subcutaneously between the scapula [60 (link)]. Minipumps contained either 0.9% saline vehicle or MitoTEMPOL (0.7 mg/kg/day; Caymen Chemical Company, Ann Arbor, MI; MT) dissolved in saline. MT combines the antioxidant moiety TEMPOL, with a lipophilic cation triphenylphosphonium [61 , 62 (link)]. Triphenylphosphonium increases mitochondrial aggregation of MT by several hundred-fold over TEMPOL alone [62 (link)–64 (link)]. MT has been observed to reduce mtOS in vitro and in vivo [64 (link), 65 (link)]. Drugs like MT are easily administered with multiple biologically active routes of administration and high blood-brain barrier permeability [66 (link)–68 (link)]. The dose of MT was chosen based on previous in vivo studies [65 (link), 69 (link), 70 (link)]. To ensure proper osmotic function as indicated by bubbling present on the surface of pumps, all pumps were incubated in a 37°C water bath for at least 24 hours prior to implantation. Surgeries were performed using aseptic techniques with isoflurane (2–3%) anesthetic.
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