Gical state [17]. Protocols of open field test were previously approved [9]. The open field test was performed 12 h after ceasing the chronic stress procedure between the second and fifth hours of the dark phase. The apparatus consisted of a rectangular area of 81 6 81 cm surrounded by a 28 cm high 1326631 wall. The area was divided into 16 squares of 20 6 20 cm by painted white lines. The field was lighted with a 40W bulb fixed 50 cm above the field. Light was focused on the center of the field with the periphery remaining dark. The mice were placed in one corner of the open field and its activity during the subsequent 5 min was assessed. Horizontal locomotion (number of times crossings of the white lines), frequency of rearing or leaning (sometimes termed vertical activity) and wall time (the time in the peripheral squares of the open field) were observed.AnimalsA total of 54 5-week-old Swiss female mice were randomly assigned to 4 groups: Control group(n = 18); stressed group(n = 18); BDNF-treated group(n = 9); BDNF-treated stressed group(n = 9). Mice were housed 9 per cage and acclimatized to the animal colony for 1 week before the start of the experimental procedures. The stress group received 30-day stress procedure. All mice received standard rodent diet and tap water ad lib under a 12 h light ark cycle (lights on 0730?930) and a constant temperature of 21?2uC and humidity of 5565 .Mouse Stressed ModelTolerance can develop when rodents are repeatedly exposed to a predictable stressor. However, this does not occur when rodents are exposed to unpredictable stress. A classic stressed model was induced by chronic unpredictable mild stress [9,14,15]. The study was conducted in compliance with Ethics Committees on Animal Research of Anhui Provincial MedChemExpress GSK126 Hospital Affiliated to Anhui Medical University. Stressors were administered once daily between 8:30 and 10:30, except the 24 h duration stressors. Stressors consisted of (1) 24 h social isolation (one mouse per cage); (2) 24 h social crowding (18 mice per cage, 32562106185 mm) plus cage tilt (cages were tilted to 30uC from the horizontal); (3) 1 h warm swim at 31uC; (4) 4 min cold swim at 8?0uC, after which they were toweled dry; (5) 5 min hot stress in oven at 42uC; (6) 24 h food deprivation; (7) 24 h water deprivation with empty drinking bottles; (8) 24 h wet cages; (9) 1 h shaker stress (160 r.p.m.); (10) 24 h light-dark shift. The different stressors were distributed randomly at an interval of 10 days. Every stressor was administered three times within 30 days.Corticosterone Enzyme ImmunoassayPlasma corticosterone was measured using a competitive enzyme immunoassay (EIA) kit (NO. 500655, Cayman Chemical Co., USA), according to manufacturer’s instructions. In brief, the samples were washed and extracted with methylene chloride; placed in wells coated with rabbit antiserum with a competitive tracer; and concentration was assessed by using a spectrophotometer (ELX800, Biotek) measuring absorbance at 412 nm and comparing samples with known dilutions.Western BlottingThe ovaries were homogenized in GSK2334470 ice-cold homogenization buffer (HB) containing 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.4), 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl2, 0.2 mM dithiothreitol, 20 mM b-glycerophosphate, 20 mM sodium 12926553 pyrophosphate, 50 mM NaF, 1 mM each of EDTA and EGTA, and protease inhibitor cocktail (11873580001, Roche, Mannheim, Germany). After the protein concentration was measured by the method of Lowry with bov.Gical state [17]. Protocols of open field test were previously approved [9]. The open field test was performed 12 h after ceasing the chronic stress procedure between the second and fifth hours of the dark phase. The apparatus consisted of a rectangular area of 81 6 81 cm surrounded by a 28 cm high 1326631 wall. The area was divided into 16 squares of 20 6 20 cm by painted white lines. The field was lighted with a 40W bulb fixed 50 cm above the field. Light was focused on the center of the field with the periphery remaining dark. The mice were placed in one corner of the open field and its activity during the subsequent 5 min was assessed. Horizontal locomotion (number of times crossings of the white lines), frequency of rearing or leaning (sometimes termed vertical activity) and wall time (the time in the peripheral squares of the open field) were observed.AnimalsA total of 54 5-week-old Swiss female mice were randomly assigned to 4 groups: Control group(n = 18); stressed group(n = 18); BDNF-treated group(n = 9); BDNF-treated stressed group(n = 9). Mice were housed 9 per cage and acclimatized to the animal colony for 1 week before the start of the experimental procedures. The stress group received 30-day stress procedure. All mice received standard rodent diet and tap water ad lib under a 12 h light ark cycle (lights on 0730?930) and a constant temperature of 21?2uC and humidity of 5565 .Mouse Stressed ModelTolerance can develop when rodents are repeatedly exposed to a predictable stressor. However, this does not occur when rodents are exposed to unpredictable stress. A classic stressed model was induced by chronic unpredictable mild stress [9,14,15]. The study was conducted in compliance with Ethics Committees on Animal Research of Anhui Provincial Hospital Affiliated to Anhui Medical University. Stressors were administered once daily between 8:30 and 10:30, except the 24 h duration stressors. Stressors consisted of (1) 24 h social isolation (one mouse per cage); (2) 24 h social crowding (18 mice per cage, 32562106185 mm) plus cage tilt (cages were tilted to 30uC from the horizontal); (3) 1 h warm swim at 31uC; (4) 4 min cold swim at 8?0uC, after which they were toweled dry; (5) 5 min hot stress in oven at 42uC; (6) 24 h food deprivation; (7) 24 h water deprivation with empty drinking bottles; (8) 24 h wet cages; (9) 1 h shaker stress (160 r.p.m.); (10) 24 h light-dark shift. The different stressors were distributed randomly at an interval of 10 days. Every stressor was administered three times within 30 days.Corticosterone Enzyme ImmunoassayPlasma corticosterone was measured using a competitive enzyme immunoassay (EIA) kit (NO. 500655, Cayman Chemical Co., USA), according to manufacturer’s instructions. In brief, the samples were washed and extracted with methylene chloride; placed in wells coated with rabbit antiserum with a competitive tracer; and concentration was assessed by using a spectrophotometer (ELX800, Biotek) measuring absorbance at 412 nm and comparing samples with known dilutions.Western BlottingThe ovaries were homogenized in ice-cold homogenization buffer (HB) containing 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.4), 100 mM KCl, 320 mM sucrose, 0.5 mM MgCl2, 0.2 mM dithiothreitol, 20 mM b-glycerophosphate, 20 mM sodium 12926553 pyrophosphate, 50 mM NaF, 1 mM each of EDTA and EGTA, and protease inhibitor cocktail (11873580001, Roche, Mannheim, Germany). After the protein concentration was measured by the method of Lowry with bov.