20, 102C112. was administered subcutaneously (subQ) at different times post-injury. We found PZ treatment preserves both synaptic and non-synaptic mitochondrial bioenergetics Etofylline at 24 hrs and that this protection is partially maintained out to 72 hrs post-injury using various dosing regimens. The results from these studies indicate that this therapeutic window for the first dose of PZ is likely within the first hour after injury, and the window for administration of the second dose seems to fall between 12 – 24 hrs. Administration of PZ was able to significantly improve mitochondrial respiration compared to vehicle-treated animals across various says of respiration for both the non-synaptic and synaptic mitochondria. The synaptic mitochondria appear to respond more robustly to PZ treatment than the non-synaptic, and further experimentation will need to be done to further understand these effects in the context of TBI. studies detailing the injection time points after the CCI-TBI and the timing of the euthanasia and tissue collection. Experimental timelines are arranged by the total length of each experiment (#s 1 ?5). Isolation of Ficoll-purified non-synaptic and synaptic cortical mitochondria Mitochondria were isolated from the ipsilateral (injured) cortex or from Sham (craniotomized, but not injured) rats as described previously (Singh et al., 2013; Singh et al., 2006b) with modifications to enable separation of synaptic and non-synaptic populations (Gilmer et al., 2010b; Patel et al., 2009). In brief, rats were deeply anesthetized with CO2, decapitated, and the cortical tissue was rapidly dissected using an 8 mm punch to collect the injury Etofylline epicenter and surrounding peri-contusional cortical tissue. Cortical tissue then was homogenized in ice-cold isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin, 20 mM HEPES, 1 mM EGTA, pH 7.2) using Potter-Elvejhem Rabbit Polyclonal to OR2L5 homogenizers. Samples were then centrifuged twice at 1,300 x rcf for 3 min at 4C. Supernatants were collected and spun at 13,000 x rcf for 10 min at 4C. The crude mitochondrial pellet was re-suspended and layered onto a Etofylline discontinuous 7.5% and 10% Ficoll gradient and centrifuged at 100,000 x rcf for 30 min at 4C. The non-synaptic mitochondria pellet was resuspended in isolation buffer without EGTA and centrifuged at 10,000 x rcf for 10 min at 4C to remove Ficoll and then re-suspended to a final concentration of approximately 10 mg/ml in isolation buffer without EGTA. The synaptosomal layer was removed from the 7.5%-10% Ficoll interface, re-suspended in isolation buffer and spun at 13,000 x rcf for 10 min at 4C to remove Ficoll. The synaptosome pellet was resuspended in isolation buffer with EGTA, placed into a nitrogen bomb at 1200 psi for 10min at 4C to release synaptic mitochondria, (Brown et al., 2004; Kristian et al., 2006) layered onto a second discontinuous 7.5% and 10% Ficoll gradient, and centrifuged at 100,000 x rcf for 30 min at 4C. The synaptic mitochondria pellet was resuspended in isolation buffer without EGTA and centrifuged at 10,000 x rcf for 10 min at 4C to remove Ficoll and resuspended to a final concentration of approximately 10 mg/ml in isolation buffer without EGTA. Protein concentrations were determined with a BCA protein assay kit (ThermoFisher Scientific, Waltham, MA) and measured at absorbance 562 nm with a BioTek Synergy HT plate reader (Winooski, VT, USA). Beginning at 48h after TBI, mitochondria obtained from individual animals were pooled based on protein yield, and therefore, each individual n for time points 48h and beyond represents 1-2 animals. Mitochondria were immediately used for respiratory analysis. Measurement of emitochondrial respiratory function Mitochondrial respiratory rates were measured using a Clarke-type electrode in a constantly stirred, sealed and thermostatically-controlled chamber (Oxytherm System, Hansatech Instruments Ltd., Norfolk, England, UK) maintained at 37C as previously described (Hill et al., 2017; Mustafa et al., 2010b; Singh et al., 2006b; Singh et al., 2007; Sullivan et al., 2003; Sullivan et al., 2004) Mitochondria loaded into the chamber.

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