Accelerated cerebromicrovascular senescence contributes to cognitive decline in a mouse model of paclitaxel (Taxol)-induced chemobrain methods
Aim. Evidence-backed execution summary for Accelerated cerebromicrovascular senescence contributes to cognitive decline in a mouse model of paclitaxel (Taxol)-induced chemobrain methods from Accelerated cerebromicrovascular senescence contributes to cognitive decline in a mouse model of paclitaxel (Taxol)-induced chemobrain.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
MATERIALS AND METHODS
reagent used in the protocol.
- Use
- Three-month-old male p16-3MR mice received PTX (5 mg/kg/day, i.p., n = 150) or vehicle (DMSO + saline, n = 50) in 2 cycles (5 days/cycle) with a 7-day interval between cycles. Mice were left to recover for 2 weeks in the original e...
Assessment of neurovascular coupling responses
reagent used in the protocol.
- Use
- NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with isoflurane (2% induction and 1% maintenance), endotracheally intubated, and ventilated. The skull was thinned and changes in cerebral blood...
Determination of senescent cell burden by flow cytometric analysis
reagent used in the protocol.
- Use
- We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al., ). In brief, single-cell suspensions were prepared. Fixed cells were stained with the RFP-Booster (AlexaFluor-488,...
Determination of senescent cell burden by flow cytometric analysis
reagent used in the protocol.
- Use
- To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surface marker CD31. First, the fraction of RFP+ senescent cells were determined as a percentage of total cells in the single cell suspensions f...
Detection of activated microglia by immunohistochemistry
reagent used in the protocol.
- Use
- Brains were perfusion-fixed and frozen OCT-embedded sagittal sections (35 µm) were cut. Sections were immunolabeled for IBA-1 (rabbit anti-mouse Iba-I antibody; 1:200, Fijifilm; overnight at 4°C) and the endothelial marker endomucin (rat monoclonal anti-mouse endom...
PTX promotes microvascular rarefaction: Protective effects of senolytic treatments
reagent used in the protocol.
- Use
- Elimination of senescent cells increases capillary density and improves endothelium-mediated neurovascular coupling responses in PTX-treated mice. (a) Segmentation of blood vessels in brain parenchyma on OCT images. Original z-stack images acquired in brains of control and PTX treated p16-3MR...
PTX impairs endothelial NO -mediated NVC responses: Protective effects of senolytic treatments
reagent used in the protocol.
- Use
- Consistent with an important role for endothelial NO in mediating functional hyperemia (Balasubramanian et al.,; Tarantini, Nyul-Toth et al., ), administration of the eNOS inhibitor L-NAME significantly decreased NVC responses in control animals ( n = 8-9) (representative...
MATERIALS AND METHODS
To identify and eliminate senescent cells, we used a novel transgenic mouse model (p16-3MR mice; Yabluchanskiy et al., ) that carries a fusion protein (3MR) under control of the p16 Ink4a promoter. 3MR contains monomeric red fluorescent protein (mRFP), which enables us to FACS-sort senescent cells...
- Use
- To identify and eliminate senescent cells, we used a novel transgenic mouse model (p16-3MR mice; Yabluchanskiy et al., ) that carries a fusion protein (3MR) under control of the p16 Ink4a promoter. 3MR contains monomeric red fluorescent protein (mRFP), which enables us to FACS-sort senescent cells...
Radial arms water maze testing
To determine how senescence induced by PTX and depletion of senescent cells affect cognitive function, spatial memory and long term memory were tested by assessing performance in the radial arms water maze at 6 months post-chemotherapy, following our published protocols (Yabluchanskiy et al., ).
- Use
- To determine how senescence induced by PTX and depletion of senescent cells affect cognitive function, spatial memory and long term memory were tested by assessing performance in the radial arms water maze at 6 months post-chemotherapy, following our published protocols (Yabluchanskiy et al., ).
Spatial memory testing of mice in Y-maze
Hippocampal-dependent contextual memory was tested with the Y-maze two-trial delayed alternation task according to our published protocol (Csiszar et al., ).
- Use
- Hippocampal-dependent contextual memory was tested with the Y-maze two-trial delayed alternation task according to our published protocol (Csiszar et al., ).
Grip strength, rotarod, and gait analysis
As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal muscle strength of mouse forelimbs. Motor coordination was assessed using an automated four-lane rotarod tool. To determine the impact o...
- Use
- As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal muscle strength of mouse forelimbs. Motor coordination was assessed using an automated four-lane rotarod tool. To determine the impact o...
Assessment of neurovascular coupling responses
NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with isoflurane (2% induction and 1% maintenance), endotracheally intubated, and ventilated. The skull was thinned and changes in cerebral blood...
- Use
- NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with isoflurane (2% induction and 1% maintenance), endotracheally intubated, and ventilated. The skull was thinned and changes in cerebral blood...
Determination of senescent cell burden by flow cytometric analysis
To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surface marker CD31. First, the fraction of RFP+ senescent cells were determined as a percentage of total cells in the single cell suspensions f...
- Use
- To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surface marker CD31. First, the fraction of RFP+ senescent cells were determined as a percentage of total cells in the single cell suspensions f...
Determination of senescent cell burden by flow cytometric analysis
Fluorescent activated cell sorting (FACS) with the low-pressure WOLF Cell Sorter™ (NanoCellect) was used to obtain a cell suspension enriched in brain senescent cells. RFP+ senescent cells were stained with cell-specific markers. Antibodies against CD31 were used to quantify the ratio of senescent...
- Use
- Fluorescent activated cell sorting (FACS) with the low-pressure WOLF Cell Sorter™ (NanoCellect) was used to obtain a cell suspension enriched in brain senescent cells. RFP+ senescent cells were stained with cell-specific markers. Antibodies against CD31 were used to quantify the ratio of senescent...
PTX induces endothelial senescence: Protective effects of senolytic treatments
To elucidate the effect of in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16-RFP+ senescent cells 6 months after PTX treatment (Figure ). PTX treatment resulted in significant increases in the number of p16-...
- Use
- To elucidate the effect of in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16-RFP+ senescent cells 6 months after PTX treatment (Figure ). PTX treatment resulted in significant increases in the number of p16-...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Depending on the experiment, statistical analyses were carried out by unpaired t test, one-way or two-way ANOVA with Fisher LSD post hoc test using GraphPad Prism 7.0, as appropriate. Differences were considered significant at p < 0.05. Data are presented as means ± standard...
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Grip strength, rotarod, and gait analysis
As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal muscle strength of mouse forelimbs. Motor coordination was assessed using an automated four-lane rotarod tool. To determine the impact of PTX treatment on gait coordination, we tested the experimental groups of mice using an automated computer assisted method (CatWalk; Noldus Information Technology Inc.) using our previously reported protocol (Ungvari et al., ).
Determination of senescent cell burden by flow cytometric analysis
We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al., ). In brief, single-cell suspensions were prepared. Fixed cells were stained with the RFP-Booster (AlexaFluor-488, 1:1000; Chromotek; US-QUO201590, 0.5 gm/L) for 30 min, centrifuged (300 × g, 10 min), and resuspended in MACS buffer (Miltenyi Biotech). The RFP-Booster allows for the detection of senescent cells that express the RFP-containing 3MR construct under the control of the p16 Ink4a promoter.
PTX promotes BBB disruption and neuroinflammation: Protective effects of senolytic treatments
Using two-photon microscopy, the relative permeability of the fluorescent tracers was measured. Figure shows background-corrected fluorescent intensity changes over time in the brain parenchyma in mice from each experimental group after the administration of different size tracers. As a measure of relative vascular permeability, the area under curve of normalized intensity changes as a function of time was calculated in each group. For the 500, 40, and 3 kDa tracers the cerebral microcirculation of PTX treated animals exhibited significantly increased permeability (Figure ). In PTX treated mice removal of senescent cells by GCV or ABT263 significantly attenuated BBB permeability ( n = 5-10), restoring it to levels observed in control mice (Figure ). This observation is consistent with the concept that endothelial senescence-...
Measurement outputs
What raw and processed outputs should exist?
As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal mu...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al.,...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surf...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
To elucidate the effect of in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16-RFP+ senescent cells 6 months after PTX treatment (Figure ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal mu...; NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with...; We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al.,...; To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surf....
from paperStatistical comparison
To elucidate the effect of in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16-RFP+ se...; Depending on the experiment, statistical analyses were carried out by unpaired t test, one-way or two-way ANOVA with Fisher LSD post hoc test using GraphPad Prism 7....; To demonstrate how PTX affects endothelial cells, samples enriched for senescent p16-RFP+ cells (Figure ) were co-sorted for the endothelial marker CD31 (Figur...; Single-cell transcriptomes were validated by quality control measures (Figure ). Unbiased Louvain clustering of cells resolved 6 robust, transcriptionally distinct c...
from paperReporting output
Report representative outputs alongside summary comparisons for As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal mu..., NVC responses were assessed as described previously (Tarantini, Balasubramanian, et al.,; Tarantini et al.,, ). In brief, mice in each group were anesthetized with..., We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al.,..., To assess senescent cell burden, a portion of the RFP-Booster-stained samples was also labelled with an antibody directed against the endothelium-specific surf....
inferred from protocolStructured statistical methods
To elucidate the effect of in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16-RFP+ se...; Depending on the experiment, statistical analyses were carried out by unpaired t test, one-way or two-way ANOVA with Fisher LSD post hoc test using GraphPad Prism 7....; To demonstrate how PTX affects endothelial cells, samples enriched for senescent p16-RFP+ cells (Figure ) were co-sorted for the endothelial marker CD31 (Figur...; Single-cell transcriptomes were validated by quality control measures (Figure ). Unbiased Louvain clustering of cells resolved 6 robust, transcriptionally distinct c...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
As an additional control, we determined how the pharmacological treatments affect muscle function in mice. To that end, the grip strength test was used to measure the maximal muscle strength of mouse forelimbs. Motor coordination was assessed using an automated four-lane rotarod tool. To determine the impact of PTX treatment on gait coordination, we tested the experimental groups of mice using an automated computer assisted method (CatWalk; Noldus Information Technology Inc.) using our previously reported protocol (Ungvari et al., ).
We used sorted cells obtained from the single-cell suspensions from the brain samples to analyze senescent cell burden using our published protocols (Yabluchanskiy et al., ). In brief, single-cell suspensions were prepared. Fixed cells were stained with the RFP-Booster (AlexaFluor-488, 1:1000; Chromotek; US-QUO201590, 0.5 gm/L) for 30 min, centrifuged (300 × g, 10 min), and resuspended in MACS buffer (Miltenyi Biotech). The RFP-Booster allows for the detection of senescent cells that express the RFP-containing 3MR construct under the control of the p16 Ink4a promoter.
Using two-photon microscopy, the relative permeability of the fluorescent tracers was measured. Figure shows background-corrected fluorescent intensity changes over time in the brain parenchyma in mice from each experimental group after the administration of different size tracers. As a measure of relative vascular permeability, the area under curve of normalized intensity changes as a function of time was calculated in each group. For the 500, 40, and 3 kDa tracers the cerebral microcirculation of PTX treated animals exhibited significantly increased permeability (Figure ). In PTX treated mice removal of senescent cells by GCV or ABT263 significantly attenuated BBB permeability ( n = 5-10), restoring it to levels observed in control mice (Figure ). This observation is consistent with the concept that endothelial senescence-related disruption of the BBB is predominantly due to the increased transcellular permeability. Leakage of plasma-derived factors through the damaged BBB can induce neuroinflammation by activating microglia (Tucsek et al., ). We found that the number of IBA1+ microglia was increased in t...
Machine-readable layer
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