Figure 1.

Study design and randomization. (A) In vitro study design. Primary cortical neuronal culture was prepared from E18 mouse brains. At 5 d in vitro (DIV), BV2-microglia cells were added to the culture and cells were pre-treated with N-acetylcysteine (NAC) or sulforaphane (SFN) monotherapy or SFN+NAC duotherapy under inflammatory conditions. Neuronal viability was assessed using microtubule-associated-protein 2 immunostaining described by [28]. Finally, secreted nitrite and TNF concentrations in the cell culture medium were assessed using Griess reagent and ELISA, respectively. (B) In vivo study design. Sham-operated experimental controls and rats with traumatic brain injury (TBI) induced with lateral fluid-percussion injury were followed-up for 2 weeks. Administration of vehicle (sterile H₂O) or NAC (500 mg/kg in sterile H₂O, i.p.) was started at 1 h post-impact. At 1 h later (i.e., at 2 h after TBI or sham-operation), rats were treated with vehicle (0.1% DMSO in PBS, pH 7.4, i.p.) or SFN (5 mg/kg in 0.1% DMSO in PBS, i.p.). NAC was administered twice a day (6 h apart) and SFN was administered once a day (between NAC injections). Neuroscore and beam walking tests were performed at baseline, and thereafter on days (D) 2, D6, and D13 post-TBI (injury on D0). Body weight was measured at D0, and then daily until the rats were killed. Tail vein blood was sampled for biomarker analysis at baseline, and on D6 and D13 post-TBI. On D14, rats were transcardially perfused for histology. (C) Randomization. A total 40 rats were included into the study. Of these, 14 animals were randomized to sham-operation and 26 to lateral fluid-percussion-induced TBI, and further, to vehicle or duotherapy treatments. One rat in the TBI group was excluded due to broken dura after the craniectomy, another rat was excluded due to a disconnected injury cap, and another due to broken dura after the impact. Acute post-impact mortality was 13%. Thus, there were 7 rats in the Sham-VEH, 7 in Sham-DUO, 10 in TBI-VEH, and 10 in TBI-DUO groups. Abbreviations: D, day; DIV, days in vitro; DMSO, dimethyl sulfoxide; DUO, duotherapy treated with N-acetylcysteine and sulforaphane; ELISA, enzyme-linked immunosorbent assay; h, hour; i.p.; intraperitoneal; NAC, N-acetylcysteine; PBS, phosphate buffered saline; RNA, ribonucleic acid; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SFN, sulforaphane; TBI, traumatic brain injury; TNF, tumor necrosis factor; VEH, vehicle; wk, week.