Stroke Model with Bioluminescence Monitoring
Objective: Measure TGF-β signaling after transient focal ischemia stroke in young and old mice using real-time bioluminescence imaging over 21 days, and determine variability in bioluminescence between individual mice and effects of estrus cycle
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Equipment1
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Materials1
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Software1
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Protocol Steps
Animal cohort assignment for variability assessment
Nine females and 5 males (5 months old) were assigned to gauge variability in bioluminescence between individual mice and determine if bioluminescence is affected by the estrus cycle
Note: This cohort was used for preliminary characterization
View evidence from paper
“Nine females and 5 males (5 months old) were used to gauge variability in bioluminescence between individual mice, and to determine if bioluminescence is affected by the estrus cycle”
Animal cohort assignment for stroke and sham surgery groups
Eighteen young females (5 months old) and eighteen old females (18 months old) were assigned to measure TGF-β signaling after stroke. Three mice from each cohort were designated for sacrifice at days 1, 3, 7, 14 and 21 after stroke. Three mice of each age group underwent sham surgery and were imaged for 21 days prior to sacrifice
Note: Experimental paradigm was repeated in young and old male mice
View evidence from paper
“Eighteen young females (5 months old) and eighteen old females (18 months old) were used to measure TGF-β signaling after stroke in real time by bioluminescence. 3 mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke”
Transient focal ischemia stroke induction
Stroke was induced in young and old female mice (and subsequently in male mice)
Note: Specific surgical technique not detailed in provided text
View evidence from paper
“Eighteen young females (5 months old) and eighteen old females (18 months old) were used to measure TGF-β signaling after stroke in real time by bioluminescence”
Sham surgery control procedure
Three mice of each age group underwent sham surgery without stroke induction
Note: Sham surgery mice served as controls for uninjured brains
View evidence from paper
“Three mice of each age group also underwent sham surgery and were imaged for 21 days prior to sacrifice”
Real-time bioluminescence imaging
Bioluminescence imaging was performed to measure TGF-β signaling in real time over 21 days post-stroke and post-sham surgery
Note: Imaging was conducted on both stroke and sham surgery groups
View evidence from paper
“measure TGF-β signaling after stroke in real time by bioluminescence”
Tissue collection at designated timepoints
Three mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke for tissue analysis
Note: Tissue was collected for immunofluorescence analysis
View evidence from paper
“3 mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke”
Immunofluorescence analysis for TGF-β localization
Localization of TGF-β1 and TGF-β signaling was determined by immunofluorescence in tissue samples from stroke and sham surgery groups
Note: Performed on sacrificed tissue samples
View evidence from paper
“3 mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke for localization of TGF-β1 and TGF-β signaling by immunoflorescence”
Repeat experimental paradigm in male mice
The entire experimental paradigm (stroke induction, sham surgery, bioluminescence imaging, and tissue collection) was repeated in young and old male mice
Note: Allows comparison of sex differences in TGF-β signaling response to stroke
View evidence from paper
“This experimental paradigm was repeated in young and old male mice”