Accelerated cerebromicrovascular senescence contributes to cognitive decline in a mouse model of paclitaxel (Taxol)-induced chemobrain methods
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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-...
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Gait recovery and locomotor function
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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...
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