Role of Interleukin-1 β in Postoperative Cognitive Dysfunction methods
Aim. Evidence-backed execution summary for Role of Interleukin-1 β in Postoperative Cognitive Dysfunction methods from Role of Interleukin-1 β in Postoperative Cognitive Dysfunction.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Quantitative Real Time Polymerase Chain Reaction
reagent used in the protocol.
- Use
- Total RNA was extracted using RNeasy Kit (Qiagen, Valencia, CA) and quantified. The 1-step quantitative real time polymer-ase chain reaction (qRT-PCR) was performed on a Rotor-Gene 6000 (Corbett Life Science, Cambridge, UK), using Assay-on-Demand premixed Taqman probe master mixes (Applied Bio-systems, Foster City,...
Cytokine Measurement
reagent used in the protocol.
- Use
- IL-6, tumor necrosis factor (TNF)- α, and IL-1 β were measured by enzyme-linked immunosorbent assay (ELISA) (Biosource, Camarillo, CA; Bender Medsystem, Burlingame, CA, respectively). Hippocampal IL-1 β was measured by ELISA (Bender Medsystem) as previously described.
Fear Conditioning Tests
Mice underwent delay or trace fear conditioning 30 minutes or 3 days prior to intervention by training with 2 trials of tone and foot-shock pairings. Three days after conditioning, mice were placed back in the original conditioning chamber, where no tone or shock was presented, to assess recall of contextual fear me...
- Use
- Mice underwent delay or trace fear conditioning 30 minutes or 3 days prior to intervention by training with 2 trials of tone and foot-shock pairings. Three days after conditioning, mice were placed back in the original conditioning chamber, where no tone or shock was presented, to assess recall of contextual fear me...
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Objective
Although postoperative cognitive dysfunction (POCD) often complicates recovery from major surgery, the pathogenic mechanisms remain unknown. We explored whether systemic inflammation, in response to surgical trauma, triggers hippocampal inflammation and subsequent memory impairment, in a mouse model of orthopedic surgery.
Methods
C57BL/6J, knock out (lacking interleukin [IL]-1 receptor, IL-1R -/- ) and wild type mice underwent surgery of the tibia under general anesthesia. Separate cohorts of animals were tested for memory function with fear conditioning tests, or euthanized at different times to assess levels of systemic and hippocampal cytokines and microglial activation; the effects of interventions, designed to interrupt inflammation (specifically and nonspecifically), were also assessed.
Interpretation
A peripheral surgery-induced innate immune response triggers an IL-1 β -mediated inflammatory process in the hippocampus that underlies memory impairment. This may represent a viable target to interrupt the pathogenesis of postoperative cognitive dysfunction.
Material and Methods
Surgery consisted of an open tibial fracture of the left hind paw with intramedullary fixation in aseptic conditions under general anesthesia with isoflurane and buprenorphine as previously described. Other than surgery, C57BL/6J mice received anesthesia/analgesia alone, or underwent surgery with concurrent administration of minocycline, enrofloxacin, or IL-1 receptor antagonist (IL-1Ra), or were not subjected to any intervention (naive). Positive controls were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS) 1mg/kg. In other experiments, IL-1R -/- and IL-1R +/+ mice were naive or subjected to surgery under anesthesia. An additional group of WT mice also received preemptive administration of IL-1Ra before surgery.
Fear Conditioning Tests
Mice underwent delay or trace fear conditioning 30 minutes or 3 days prior to intervention by training with 2 trials of tone and foot-shock pairings. Three days after conditioning, mice were placed back in the original conditioning chamber, where no tone or shock was presented, to assess recall of contextual fear memory. After 3 hours, mice were placed in a novel environment (different context from training) to test for auditory-cued memory. Our measure of associative learning was freezing, a common and reliable index of learning and memory in fear conditioning preparations.
Social Olfactory Discrimination Task
This test consisted of 4,2-minute-long exposures, with 10-minute intervals, of the test mouse to a stimulus mouse in a neutral cage. At the 5th trial, a new novel mouse stimulus was presented. After a retention interval of 24 hours, animals undergoing testing were re-exposed to the previously presented stimulus mouse and subsequently to an additional new stimulus mouse. A trained observer, blinded to the treatment, measured the duration of the investigatory behavior of each stimulus animal separately.
Cytokines Are Increased in the Hippocampus after Surgery
Hippocampal IL-1 β and IL-6 transcription increased from baseline (Kruskal-Wallis; IL-1 β: H = 12.35, p = 0.0063; IL-6: H = 16.61; p = 0.0008) 2-fold ( p < 0.01) and 4-fold ( p < 0.001), respectively, 6 hours after surgery ( ). Consistently, when the expression of hippocampal IL-1 β was assessed 6 hours after surgery (ANOVA; F 4,30 = 5.906, p = 0.0013), there was a 2-fold increase of IL-1 β compared to naive counterparts (5.53 ± 0.89 vs 2.73 ± 0.39pg/100 µ g of proteins, p < 0.05; see ). IL-1 β expression in the hippocampus was not changed from baseline in animals exposed to anesthesia alone. Minocycline, but not enrofloxacin, reduced IL-1 β expression in surgical animals to naive levels.
Surgery Induces Reactive Microgliosis in the Hippocampus
Surgery induced significant morphological changes of microglial reactivity at 24 hours (Kruskal-Wallis: H = 14.63, p = 0.0022; n = 7; ) compared to naive and animals treated only with anesthesia ( p < 0.01). Surgery-induced reactive microgliosis was still higher than baseline ( p < 0.05) at day 3 (H = 10.29, p = 0.0163) and returned to normal by 7 days (H = 1.56, p = 0.6686). Minocycline prevented postoperative reactive microgliosis.
Measurement outputs
What raw and processed outputs should exist?
Total RNA was extracted using RNeasy Kit (Qiagen, Valencia, CA) and quantified. The 1-step quantitative real time polymer-ase chain reaction (qRT-PCR) was performed on a Rotor-G...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
IL-6, tumor necrosis factor (TNF)- α, and IL-1 β were measured by enzyme-linked immunosorbent assay (ELISA) (Biosource, Camarillo, CA; Bender Medsystem, Burlingame, C...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
This test consisted of 4,2-minute-long exposures, with 10-minute intervals, of the test mouse to a stimulus mouse in a neutral cage. At the 5th trial, a new novel mouse stimulus...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Data are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Data are expressed as mean ± standard error of the mean.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Total RNA was extracted using RNeasy Kit (Qiagen, Valencia, CA) and quantified. The 1-step quantitative real time polymer-ase chain reaction (qRT-PCR) was performed on a Rotor-G...; IL-6, tumor necrosis factor (TNF)- α, and IL-1 β were measured by enzyme-linked immunosorbent assay (ELISA) (Biosource, Camarillo, CA; Bender Medsystem, Burlingame, C...; This test consisted of 4,2-minute-long exposures, with 10-minute intervals, of the test mouse to a stimulus mouse in a neutral cage. At the 5th trial, a new novel mouse stimulus...; Data are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple....
from paperStatistical comparison
Data are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple...; Hippocampal IL-1 β and IL-6 transcription increased from baseline (Kruskal-Wallis; IL-1 β: H = 12.35, p = 0.0063; IL-6: H = 16.61; p = 0.0008) 2-fold ( p < 0.01) and...; Surgery induced significant morphological changes of microglial reactivity at 24 hours (Kruskal-Wallis: H = 14.63, p = 0.0022; n = 7; ) compared to naive and animals treated onl...; As expected, there was no difference in freezing time between the groups during training (data not shown). Surgery performed 30 minutes following training significantly reduced...
from paperReporting output
Report representative outputs alongside summary comparisons for Total RNA was extracted using RNeasy Kit (Qiagen, Valencia, CA) and quantified. The 1-step quantitative real time polymer-ase chain reaction (qRT-PCR) was performed on a Rotor-G..., IL-6, tumor necrosis factor (TNF)- α, and IL-1 β were measured by enzyme-linked immunosorbent assay (ELISA) (Biosource, Camarillo, CA; Bender Medsystem, Burlingame, C..., This test consisted of 4,2-minute-long exposures, with 10-minute intervals, of the test mouse to a stimulus mouse in a neutral cage. At the 5th trial, a new novel mouse stimulus..., Data are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple....
inferred from protocolStructured statistical methods
Data are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple...; Hippocampal IL-1 β and IL-6 transcription increased from baseline (Kruskal-Wallis; IL-1 β: H = 12.35, p = 0.0063; IL-6: H = 16.61; p = 0.0008) 2-fold ( p < 0.01) and...; Surgery induced significant morphological changes of microglial reactivity at 24 hours (Kruskal-Wallis: H = 14.63, p = 0.0022; n = 7; ) compared to naive and animals treated onl...; As expected, there was no difference in freezing time between the groups during training (data not shown). Surgery performed 30 minutes following training significantly reduced...
source structuredSource and audit
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Evidence quotes (8)
Although postoperative cognitive dysfunction (POCD) often complicates recovery from major surgery, the pathogenic mechanisms remain unknown. We explored whether systemic inflammation, in response to surgical trauma, triggers hippocampal inflammation and subsequent memory impairment, in a mouse model of orthopedic surgery.
C57BL/6J, knock out (lacking interleukin [IL]-1 receptor, IL-1R -/- ) and wild type mice underwent surgery of the tibia under general anesthesia. Separate cohorts of animals were tested for memory function with fear conditioning tests, or euthanized at different times to assess levels of systemic and hippocampal cytokines and microglial activation; the effects of interventions, designed to interrupt inflammation (specifically and nonspecifically), were also assessed.
A peripheral surgery-induced innate immune response triggers an IL-1 β -mediated inflammatory process in the hippocampus that underlies memory impairment. This may represent a viable target to interrupt the pathogenesis of postoperative cognitive dysfunction.
Surgery consisted of an open tibial fracture of the left hind paw with intramedullary fixation in aseptic conditions under general anesthesia with isoflurane and buprenorphine as previously described. Other than surgery, C57BL/6J mice received anesthesia/analgesia alone, or underwent surgery with concurrent administration of minocycline, enrofloxacin, or IL-1 receptor antagonist (IL-1Ra), or were not subjected to any intervention (naive). Positive controls were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS) 1mg/kg. In other experiments, IL-1R -/- and IL-1R +/+ mice were naive or subjected to surgery under anesthesia. An additional group of WT mice also received preemptive administration of IL-1Ra before surgery.
Mice underwent delay or trace fear conditioning 30 minutes or 3 days prior to intervention by training with 2 trials of tone and foot-shock pairings. Three days after conditioning, mice were placed back in the original conditioning chamber, where no tone or shock was presented, to assess recall of contextual fear memory. After 3 hours, mice were placed in a novel environment (different context from training) to test for auditory-cued memory. Our measure of associative learning was freezing, a common and reliable index of learning and memory in fear conditioning preparations.
This test consisted of 4,2-minute-long exposures, with 10-minute intervals, of the test mouse to a stimulus mouse in a neutral cage. At the 5th trial, a new novel mouse stimulus was presented. After a retention interval of 24 hours, animals undergoing testing were re-exposed to the previously presented stimulus mouse and subsequently to an additional new stimulus mouse. A trained observer, blinded to the treatment, measured the duration of the investigatory behavior of each stimulus animal separately.
Hippocampal IL-1 β and IL-6 transcription increased from baseline (Kruskal-Wallis; IL-1 β: H = 12.35, p = 0.0063; IL-6: H = 16.61; p = 0.0008) 2-fold ( p < 0.01) and 4-fold ( p < 0.001), respectively, 6 hours after surgery ( ). Consistently, when the expression of hippocampal IL-1 β was assessed 6 hours after surgery (ANOVA; F 4,30 = 5.906, p = 0.0013), there was a 2-fold increase of IL-1 β compared to naive counterparts (5.53 ± 0.89 vs 2.73 ± 0.39pg/100 µ g of proteins, p < 0.05; see ). IL-1 β expression in the hippocampus was not changed from baseline in animals exposed to anesthesia alone. Minocycline, but not enrofloxacin, reduced IL-1 β expression in surgical animals to naive levels.
Surgery induced significant morphological changes of microglial reactivity at 24 hours (Kruskal-Wallis: H = 14.63, p = 0.0022; n = 7; ) compared to naive and animals treated only with anesthesia ( p < 0.01). Surgery-induced reactive microgliosis was still higher than baseline ( p < 0.05) at day 3 (H = 10.29, p = 0.0163) and returned to normal by 7 days (H = 1.56, p = 0.6686). Minocycline prevented postoperative reactive microgliosis.
Machine-readable layer
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