Mitochondrial OCR measurement

The MCAs from IPSI, CONTRA, Sham operated, and Naïve rats were isolated from the hemispheres 48 ± 6 h after tMCAO surgery. Mitochondrial OCR was measured by the Seahorse Bioscience XFe24 Analyzer.¹³ Arteries were placed in wells of a 24-well islet plate (#101122-100, Agilent Technologies, Santa Clara, CA), each containing 525 µl of Seahorse XF Assay medium (#102365-100, Seahorse Bioscience, 5.0 mmol/l glucose and 2.0 mmol/l pyruvate, pH 7.4 and 37℃). Our assay consisted of eight cycles for baseline measurements, a media injection after the third measurement, and five sequentially injected cycles of oligomycin (2 µmol/l), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µmol/l), and 1.5 µmol/l each of antimycin and rotenone. The protein concentration of each well was determined by Pierce BCA protein assay and used to normalize the raw OCR values by Wave 2.3.0 Software, then exported to GraphPad and Excel which calculated the components of mitochondrial respiration, as described previously. The OCR values were expressed as pmol/minute/µg protein. A schematic illustration is presented in Figure 3(a).

Mitochondrial respiration of MCAs. (a) Schematic of the Seahorse experimental design, showing sequence of agent injections to elicit different components of mitochondrial respiration. (b) Continuous tracings of protein normalized mitochondrial oxygen consumption rate expressed in pmol/min/µg protein unit in each group. Calculated values of mitochondrial OCR with statistical analyses shown for (c) non-mitochondrial respiration, (d) basal respiration, (e) proton leak, (f) ATP production, (g) maximal respiration, and H) spare respiratory capacity in IPSI, CONTRA, Sham, and Naïve MCAs. Data are expressed as mean ± SEM. *IPSI vs. NAÏVE, P < 0.05; #NAÏVE vs. SHAM, P < 0.05; $IPSI vs. SHAM and NAÏVE, CONTRA vs. NAIVE, P < 0.05; &IPSI vs. CONTRA, SHAM, and NAÏVE, P < 0.05; @CONTRA vs. SHAM, P < 0.05.