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Protocol Steps
1
Animal treatment initiation
Begin valganciclovir treatment of transgenic v-WT and v-TK mice at 8 weeks of age
12 weeks for behavioral testing cohortNot specified
Note: Different treatment durations used for different experimental cohorts (8 weeks for dexamethasone, 10-19 weeks for endocrine, 12 weeks for behavior, 4 weeks for histology)
View evidence from paper
“Male v-WT and v-TK mice were treated with valganciclovir for 8 weeks (dexamethasone experiment), 10-19 weeks (endocrine), 12 weeks (behavior) or 4 weeks (histology), beginning at 8 weeks of age”
2
Irradiation of control mice
Irradiate male C57Bl/6 mice under pentobarbital anesthesia
Not specifiedNot specified
Note: Control group; tested 9 weeks after irradiation
View evidence from paper
“Male C57Bl/6 mice were irradiated under pentobarbital anesthesia, as described previously, and tested 9 weeks later”
3
Dexamethasone suppression test preparation
Inject dexamethasone (50 μg/kg in propylene glycol) or vehicle 90 minutes prior to restraint stress
90 minutes before restraintNot specified
Note: Dexamethasone suppression test to assess HPA axis function
View evidence from paper
“For the dexamethasone suppression test, dexamethasone (Sigma; 50 μg/kg in propylene glycol) or vehicle were injected 90 min prior to restraint”
4
Restraint stress application
Apply restraint stress to mice for 10 minutes
10 minutesNot specified
Note: Used for dexamethasone suppression test and as a stressor condition
View evidence from paper
“blood was sampled immediately following 10 min restraint”
5
Blood sampling for corticosterone measurement
Obtain submandibular blood samples from mice under different conditions: home cage, novel box exploration, restraint, or isoflurane exposure
Not specifiedNot specified
Note: Samples collected directly from home cage or after various stressor exposures
View evidence from paper
“Serum corticosterone was measured by radioimmunoassay (MP Biomedicals) from submandibular blood samples obtained directly from the home cage condition or after exploration of a novel box, restraint, or isoflurane exposure”
6
Corticosterone measurement
Measure serum corticosterone levels using radioimmunoassay
Not specifiedNot specified
Note: Performed by MP Biomedicals
View evidence from paper
“Serum corticosterone was measured by radioimmunoassay (MP Biomedicals)”
7
Behavioral testing - Novelty Suppressed Feeding test
Conduct NSF test on different cohorts of mice following 30 minutes of restraint or directly from home cage
30 minutes restraint prior to test, or direct from home cageNot specified
Note: Different cohorts tested in NSF test, elevated plus maze, forced swim test, and sucrose preference test
View evidence from paper
“Behavioral tests were performed following 30 min of restraint or directly from the home cage. Different cohorts of mice were tested in the NSF test, elevated plus maze, forced swim test and sucrose preference test as previously described”
8
BrdU administration for immunohistochemistry
Administer BrdU to mice 6 weeks prior to sacrifice (for PVN analysis) or 24 hours prior to sacrifice
6 weeks or 24 hours before sacrificeNot specified
Note: Timing varies depending on brain region being analyzed
View evidence from paper
“For immunohistochemical analyses, mice were given BrdU 6 weeks (for PVN analysis) or 24 hours prior to sacrifice”
9
Brain tissue preparation and immunostaining
Prepare brain sections and perform immunostaining for BrdU-labeled cells
Not specifiedNot specified
Note: Performed as previously described
View evidence from paper
“brain sections were immunostained as previously described, and labeled cells were counted stereologically”
10
Stereological cell counting
Count BrdU-labeled cells stereologically in brain sections
Not specifiedNot specified
Note: Quantification of cell proliferation in specific brain regions
View evidence from paper
“labeled cells were counted stereologically”
Subjects / Specimens
Species
mouse
Strain
C57Bl/6:CD-1 mixed background, with transgenic v-WT and v-TK mice
Age
8 weeks at treatment start; tested at various timepoints (12 weeks treatment duration for behavior)
Sex
male
Count
Not specified
Weight
Not specified
Transgenic mice expressing HSV-TK under the GFAP promoter; different cohorts tested in different behavioral tests