Dynamics and Correlation of Serum Cortisol and Corticosterone under Different Physiological or Stressful Conditions in Mice methods
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Materials and Methods
Mouse care and use were conducted exactly in accordance with the guidelines and approved by the Animal Research Committee of the Shandong Agricultural University, P. R. China (Permit number: 20010510). According to the guidelines of the committee, the animal handling staff (including each post-doc, doctoral or masters student) must be trained before using animals. Mice must be housed in a temperature-controlled room with proper darkness-light cycles, fed with a regular diet, and maintained under the care of the Experimental Animal Center, Shandong Agricultural University College of Animal Science and Vet Medicine. In the present study, mice were sacrificed by decapitation. The only procedure performed on the dead animals was the collection of trunk blood.
Mice
Mice of the Kunming strain were used at the age of 6-8 weeks. The mice were kept in a room with 14h/10h light-dark cycles, the dark starting from 20:00 pm. The stage of the estrous cycle in female mice was determined by observing vaginal appearance [ ] between 19:30 and 20:00 pm on each experimental day. Mice at different stages of the estrous cycle were then sacrificed to collect blood. Immediately following blood collection, the mice were examined again by observing vaginal lavage smears [ ] to confirm their estrous cycle stages. Only blood collected from mice at the desired stage of the estrous cycle was used for hormone assays.
Procedures for restraint stress
Restraint of small animals is an experimental procedure developed for studying psychogenic stress [, ]. According to Golub et al. [ ], psychogenic implies that no invasive physical procedure or tissue trauma is involved but, rather, that the stress response is initiated in the brain by the psychological distress of being unable to move freely. To meet these requirements, we have established a new restraint system in which an individual mouse was put in a micro-cage, constructed by the authors, which was placed in an ordinary home cage. The oblong micro-cage was made of a steel-wire screen and measured 10 cm in width and 2 cm in height. The micro-cage offered the same photoperiod and controlled temperature as in the home cage for the unstressed animals. While in the micro-cage, mice could move back and forth to some extent and could take food and water freely, although they could not...
Procedures for forced swimming stress
For forced swimming testing, animals were forced to swim for different times in a rectangular plastic tank (45 × 35 × 18 cm) containing 15 cm deep water. The water temperature was maintained at approximately 23°C. After swimming, the mice were toweled dry before being sacrificed for blood collection.
Procedures for unpredictable stress
For unpredictable stress treatment, mice were exposed to different stressors for two rounds of 4 days. Stressors used on different days were as follows: Day 1, 2-h heat stress in oven at 42°C; Day 2, 2-h shaker stress (160 rpm); Day 3, 2-h forced swimming at 23°C; and Day 4, 8-h restraint stress. Whereas the stressors lasting for 2 h were administered from 14:00 to 16:00 pm, the 8-h restraint took place from 8:00 am to 16:00 pm as described above.
Preparation of blood serum
In the present study, mice were always sacrificed at 15:00-16:00 pm for blood collection except for the experiment on the effect of the estrous cycle stage in which mice were always killed at 19:30-20:00 pm and for the experiment on the effect of circadian rhythm in which animals were killed at different times of day. Mice were decapitated rapidly at the end of the stress period, and trunk blood (about 1 ml) was collected into ice-cooled centrifugal tubes and centrifuged (1700 × g, 10 min, 4°C) to separate serum. The serum collected was divided into two parts; one for assay of cortisol and the other for assay of corticosterone. The serum samples were stored at -80°C until hormone assay.
Hormone assays
Radioimmunoassay for cortisol was conducted by the Central Hospital of Tai-An City using commercial kits from 3V Biomedical Techniques Co. Ltd., Weifang, China. The kit measures total cortisol in serum including the cortisol combined with corticosteroid-binding globulin (CBG). The minimum level of detection for assays of cortisol was 0.15 ng/ml. The intra- and inter-assay coefficients of variation were <10% and <10%. The cross reactivity of the cortisol RIA kit for corticosterone is 0.11% (tested at the 50% binding). To further evaluate the cross-reactivity of the cortisol RIA with corticosterone, corticosterone and cortisol standards or their mixtures at different concentrations were subjected to the radioimmunoassay kit.
Hormone assays
Corticosterone concentrations were measured with an ELISA kit purchased from Arbor Assays Company (Catalog Number K014-H1). The minimum level of detection of the kit was 16.9 pg/ml corticosterone. The kit measures total corticosterone in serum including the corticosterone combined with corticosteroid-binding globulin (CBG). The cross reactivity of the corticosterone kit for cortisol is 0.38% (tested at the 50% binding). Briefly, 50 µl of standards or samples were added in duplicate to wells of the micro-titer plate. 75-µl of assay buffer was added to the non-specific binding (NSB) wells and 50 µl of assay buffer was added to wells to act as maximum binding wells. Then, 25 µl of the DetectX Corticosterone Conjugate and 25 µl of the DetectX Corticosterone Antibody (except the NSB wells) were added to each well and the titer plate was shaken for 1 h at room tempe...
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Evidence quotes (8)
Mouse care and use were conducted exactly in accordance with the guidelines and approved by the Animal Research Committee of the Shandong Agricultural University, P. R. China (Permit number: 20010510). According to the guidelines of the committee, the animal handling staff (including each post-doc, doctoral or masters student) must be trained before using animals. Mice must be housed in a temperature-controlled room with proper darkness-light cycles, fed with a regular diet, and maintained under the care of the Experimental Animal Center, Shandong Agricultural University College of Animal Science and Vet Medicine. In the present study, mice were sacrificed by decapitation. The only procedure performed on the dead animals was the collection of trunk blood.
Mice of the Kunming strain were used at the age of 6-8 weeks. The mice were kept in a room with 14h/10h light-dark cycles, the dark starting from 20:00 pm. The stage of the estrous cycle in female mice was determined by observing vaginal appearance [ ] between 19:30 and 20:00 pm on each experimental day. Mice at different stages of the estrous cycle were then sacrificed to collect blood. Immediately following blood collection, the mice were examined again by observing vaginal lavage smears [ ] to confirm their estrous cycle stages. Only blood collected from mice at the desired stage of the estrous cycle was used for hormone assays.
Restraint of small animals is an experimental procedure developed for studying psychogenic stress [, ]. According to Golub et al. [ ], psychogenic implies that no invasive physical procedure or tissue trauma is involved but, rather, that the stress response is initiated in the brain by the psychological distress of being unable to move freely. To meet these requirements, we have established a new restraint system in which an individual mouse was put in a micro-cage, constructed by the authors, which was placed in an ordinary home cage. The oblong micro-cage was made of a steel-wire screen and measured 10 cm in width and 2 cm in height. The micro-cage offered the same photoperiod and controlled temperature as in the home cage for the unstressed animals. While in the micro-cage, mice could move back and forth to some extent and could take food and water freely, although they could not turn around. Observations in our laboratory showed that the average intake of food and water did not differ significantly between unstressed control mice and mice restrained for different times using our restraint system (data not shown). Thus, mice restrained in this system did not suffer from any...
For forced swimming testing, animals were forced to swim for different times in a rectangular plastic tank (45 × 35 × 18 cm) containing 15 cm deep water. The water temperature was maintained at approximately 23°C. After swimming, the mice were toweled dry before being sacrificed for blood collection.
For unpredictable stress treatment, mice were exposed to different stressors for two rounds of 4 days. Stressors used on different days were as follows: Day 1, 2-h heat stress in oven at 42°C; Day 2, 2-h shaker stress (160 rpm); Day 3, 2-h forced swimming at 23°C; and Day 4, 8-h restraint stress. Whereas the stressors lasting for 2 h were administered from 14:00 to 16:00 pm, the 8-h restraint took place from 8:00 am to 16:00 pm as described above.
In the present study, mice were always sacrificed at 15:00-16:00 pm for blood collection except for the experiment on the effect of the estrous cycle stage in which mice were always killed at 19:30-20:00 pm and for the experiment on the effect of circadian rhythm in which animals were killed at different times of day. Mice were decapitated rapidly at the end of the stress period, and trunk blood (about 1 ml) was collected into ice-cooled centrifugal tubes and centrifuged (1700 × g, 10 min, 4°C) to separate serum. The serum collected was divided into two parts; one for assay of cortisol and the other for assay of corticosterone. The serum samples were stored at -80°C until hormone assay.
Radioimmunoassay for cortisol was conducted by the Central Hospital of Tai-An City using commercial kits from 3V Biomedical Techniques Co. Ltd., Weifang, China. The kit measures total cortisol in serum including the cortisol combined with corticosteroid-binding globulin (CBG). The minimum level of detection for assays of cortisol was 0.15 ng/ml. The intra- and inter-assay coefficients of variation were <10% and <10%. The cross reactivity of the cortisol RIA kit for corticosterone is 0.11% (tested at the 50% binding). To further evaluate the cross-reactivity of the cortisol RIA with corticosterone, corticosterone and cortisol standards or their mixtures at different concentrations were subjected to the radioimmunoassay kit.
Corticosterone concentrations were measured with an ELISA kit purchased from Arbor Assays Company (Catalog Number K014-H1). The minimum level of detection of the kit was 16.9 pg/ml corticosterone. The kit measures total corticosterone in serum including the corticosterone combined with corticosteroid-binding globulin (CBG). The cross reactivity of the corticosterone kit for cortisol is 0.38% (tested at the 50% binding). Briefly, 50 µl of standards or samples were added in duplicate to wells of the micro-titer plate. 75-µl of assay buffer was added to the non-specific binding (NSB) wells and 50 µl of assay buffer was added to wells to act as maximum binding wells. Then, 25 µl of the DetectX Corticosterone Conjugate and 25 µl of the DetectX Corticosterone Antibody (except the NSB wells) were added to each well and the titer plate was shaken for 1 h at room temperature. After the plate was washed using the wash solution and blot dried by hitting plate onto paper towels, 100 µl of the TMB Substrate were added to each well and incubated for 30 min at room temperature. The optical density (O.D.) of corticosterone was read at 450 nm wavelength using a plat...
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