Astaxanthin

Carotenoids were extracted as described above and analyzed using high-performance liquid chromatography (1260 Infinity II, Agilent, CA, USA) equipped with a C30 column (YMC Carotenoid column, 250 mm, 5 μm pore size). Mobile phase A consisted of 15:81:4 Methyl tert-Butyl Ether (MTBE):methanol:water by volume, and mobile phase B consisted of 81:15:4MTBE: methanol:water by volume. Using a flow rate of 1.0 mL/min at 20 °C, a linear elution gradient from 100% A to 100% B over 15 min was followed by 12 min of 100% B before returning to mobile phase A over 3 min. HPLC standards (astaxanthin, lycopene, β-carotene, zeaxanthin, and canthaxanthin) were purchased from Santa Cruz Biotechnology for identification of carotenoid retention times. zeaxanthin was used to identify isozeaxanthin as this compound cannot be purchased, and these isomers are known to co-elute using C18 chromatography [20 (link)].

Mice were randomly assigned to sham, vehicle-treated, and Astaxanthin-treated groups. Astaxanthin (Santa Cruz Biotechnology, USA, 98 % pure) was dissolved in corn oil (1 mL/kg) immediately before use. Thirty minutes after induction of CCI, 4 doses (10, 25, 50, or 100 mg/kg body weight) of Astaxanthin were administered via intraperitoneal injection. The doses of Astaxanthin used here followed the doses used by Zheng et al. [22 (link)] with some modification. Control mice were treated with an intraperitoneal injection of an equal volume of corn oil 30 min after CCI.
To further elucidate the relationship between NKCC1 and AQP4 in TBI-induced brain edema, the NKCC1-specific inhibitor bumetanide (BU) (Sigma-Aldrich, St. Louis, MO, USA) was administered intravenously (10 mg/kg body weight, as previous described [23 (link)]) 20 min before CCI. As a control, mice received intravenous injections of equal volumes of saline solution.

For carotenoid quantification, two methods were used, total carotenoid determination using spectrophotometry or liquid chromatography combined with a diode array detector. In order to determine the carotenoid content, two 1 mL samples were taken from each flask at the indicated time after induction. Samples were stored in amber microtubes to prevent photodegradation. The cell pellet was collected by 12,000× g for 1 min. One pellet was lyophilized and weighed to obtain the cell dry weight. The other was extracted with 1 mL of 1:1 (v:v) ethanol-acetone solution. The samples were vortexed to mix and were incubated in the dark for 1 h at room temperature. The samples were centrifuged again at 12,000× g for 1 min, and 200 μL of the liquid phase was transferred to a 96-well plate, and absorbance was measured using a BioTek Synergy 4 (Agilent, CA, USA) plate reader at 475 nm. Astaxanthin was purchased from Santa Cruz Biotechnologies (Dallas, TX, USA) and used to create a standard curve and was used as a proxy for total carotenoids. Total carotenoids were calculated using the following equation: $$Total Carotenoids \left(\mathsf{\mu }\mathrm{g}/\mathrm{g}\right)=\frac{Abs-blank}{0.0799 \mathrm{m}\mathrm{L}/\mathsf{\mu }\mathrm{g}}÷dry cell weight (\mathrm{g}/\mathrm{m}\mathrm{L})$$