P3972

Progesterone (P3972, Sigma) was prepared in sterile 30% 2-hydroxypropyl-beta-cyclodextrin (HBC; H107, Sigma) solution for in vivo experiments. After confirming tumor establishment, mice were randomly divided into three groups (n = 16 each): Vehicle (HBC); progesterone (8 mg/kg, PROG 8); and progesterone (100 mg/kg, PROG 100). An additional group (n = 8) of untreated, non-tumor-bearing nude mice served as controls to assess drug toxicity based on spontaneous locomotor activity, survival, and body weight. A s.c. injection of either progesterone or vehicle alone was given daily at the same approximate time for 28 days. On day 29, all vehicle-treated mice were euthanized because they showed the maximum permitted tumor burden according to Emory IACUC guidelines. Eight mice from each progesterone-treated group were euthanized along with the vehicle group. The remaining 8 mice/group were kept alive for survival studies. The endpoint criterion for the survival study was the maximum tumor burden allowed by the IACUC of Emory University. These animals continued to receive progesterone injections until they reached the tumor burden limit. They were then imaged for tumor volume using BLI.

After 7 days of acclimatization, animals were randomly assigned to the following treatment groups: (1) Sham (n = 24), unstressed controls given vehicle; (2) Sham+PROG (n = 24), unstressed controls given PROG; (3) Stress+Sham (n = 24), stressed controls given vehicle; (4) Stress+Sham+PROG (n = 24), stressed controls given PROG; (5) ISCH (n = 24), unstressed animals subjected to global ischemia given vehicle; (6) ISCH+PROG (n = 24), unstressed animals subjected to global ischemia given PROG; (7) Stress+ISCH (n = 24), stressed animals subjected to global ischemia given vehicle; and (8) Stress+ISCH+PROG (n = 24), stressed animals subjected to global ischemia given PROG. PROG (8 mg/kg/b.w.; P3972, Sigma, St. Louis, MO, USA) or vehicle (20% 2-hydroxypropyl b-cyclodextrin in sterile water; H107, Sigma, St. Louis, MO, USA) were administered by intraperitoneal injection at 2 h post post-ischemia followed by subcutaneous injections at 6 h, and once every 24 h post-injury for 5 days, and then for 2 days with progressively halved dosages. This dose of PROG has effectively produced functional improvements and reduced ischemic injury severity [92 (link),93 (link)]. PROG and vehicle were prepared and coded in advance, thus that investigators administering these solutions were blinded to their identity.