✍🏼 Rafal Gulej, Ádám Nyúl-Tóth, Chetan Ahire, Jordan DelFavero, Priya Balasubramanian, Tamas Kiss, Stefano Tarantini, Zoltan Benyo, Pal Pacher, Boglarka Csik, Andriy Yabluchanskiy, Peter Mukli, Anna Kuan-Celarier, István A Krizbai, Judith Campisi, William E. Sonntag, Anna Csiszar, Zoltan Ungvari

🏠 Department of Neurosurgery, University of Oklahoma Health Sciences Center, Oklahoma City, OK

📑 GeroScience (2023)

Abstract
Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI.

How the WOLF was used in this study
The authors used the WOLF Cell Sorter to enrich viable cells from irradiated mouse brain tissues prior to downstream analyses. After isolating brain cells and staining them with a viability dye (SYTOX™ Green) to distinguish live from dead cells, they removed dead cells and debris, isolating a high-quality live cell suspension. This gentle sorting approach preserved cell integrity and RNA quality, making the enriched live cells suitable for subsequent transcriptomic studies aimed at understanding how senolytic treatment with Navitoclax impacts cellular senescence and blood-brain barrier function following irradiation.