Radial Arms Water Maze and Neurovascular Assessment in PTX-Induced Chemobrain

Aging Cell article metadata identifies DOI 10.1111/acel.13832, PMCID PMC10352561, PMID 37243381, and the source title.

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 environment. Then, PTX treated mice were assigned randomly to three groups. Two groups received the senolytic drug ABT263 (Navitoclax, Chemgood, C-1009, i.p., 1.5 mg/kg/daily in DMSO/saline) or ganciclovir (GCV [TSZCHEM, RG001, >99%]; i.p. 25 mg/kg/daily) for 5 days and 2 cycles with a 2 week interval between cycles. At the end of 6 months after the PTX protocol, mice were tested for cognitive functions, NVC responses, and microvascular density and BBB integrity, and then were euthanized for tissue collection.

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.

Hippocampal-dependent contextual memory was tested with the Y-maze two-trial delayed alternation task according to our published protocol.

Grip strength 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, experimental groups were tested using an automated computer assisted method (CatWalk; Noldus Information Technology Inc.) using a previously reported protocol.

To assess BBB permeability and cerebromicrovascular density, mice were equipped with a chronic cranial window and intravital two-photon microscopy-based and optical coherence tomography (OCT) based imaging methods were used as previously described.

Mice were anesthetized with isoflurane (2% induction and 1% maintenance), endotracheally intubated, and ventilated. The skull was thinned and changes in cerebral blood flow (CBF) were assessed above the left somatosensory whisker barrel cortex in response to mechanical stimulation of the right whiskers. CBF responses were repeated after administering L-NAME. At the end of the experiments, animals were transcardially perfused with ice-cold PBS and decapitated.

Fixed cells were stained with the RFP-Booster (AlexaFluor-488, 1:1000; Chromotek; US-QUO201590, 0.5 gm/L) for 30 min, centrifuged (300 x g, 10 min), and resuspended in MACS buffer (Miltenyi Biotech). RFP+ senescent cells were determined using a Guava EasyCyte BGR HT Flow Cytometer (Luminex). Fluorescent activated cell sorting with the low-pressure WOLF Cell Sorter (NanoCellect) was used to obtain a cell suspension enriched in brain senescent cells.

A single-cell transcriptomics-based method was used. The study used a gel bead-in-emulsion-based droplet sequencing method and identified cerebromicrovascular endothelial cells and other brain cell types on the basis of their gene expression profile.

Spatial transcriptomics was used to assess spatial distribution of senescent cells in brains of PTX-treated mice.

Sections were immunolabeled for IBA-1 (rabbit anti-mouse Iba-I antibody; 1:200, Fujifilm; overnight at 4 C) and the endothelial marker endomucin (rat monoclonal anti-mouse endomucin antibody; 1:50, Invitrogen; overnight at 4 C). Confocal images were obtained using Leica SP8 MP confocal laser scanning microscope.

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.