Up to 90% of brain cancer survivors experience radiation-induced damage to the blood-brain barrier (BBB), as well as neuronal and bone loss. Research suggests that radiation increases mitochondrial reactive oxygen species (mtROS), leading to mitochondrial DNA (mtDNA) damage and its release as cell-free DNA. This process activates Toll-like receptor 9 (TLR9), triggering an immune response and further ROS production. Given that TLR9 is also implicated in bone metabolism, this study investigates the impact of endothelial-specific TLR9 deletion on BBB permeability, cognitive function, and bone health following cranial irradiation.

To explore this, endothelial-specific TLR9 knockout (eTLR9⁻/⁻) mice were exposed to cranial irradiation (12 Gy, X-ray). After 30 days, memory and learning abilities were assessed using the Novel Object Recognition test, which revealed significant cognitive impairment in wild-type (WT) mice, whereas eTLR9⁻/⁻ mice exhibited improved performance. BBB integrity was evaluated through immunohistochemistry with anti-mouse IgG, showing markedly higher permeability in WT mice compared to eTLR9⁻/⁻ mice. Surprisingly, bone density measurements using µCT scans demonstrated significantly lower bone density in eTLR9⁻/⁻ mice, indicating greater bone loss in the knockout group.

These findings suggest that endothelial TLR9 deletion provides neuroprotection by mitigating BBB disruption and cognitive decline after radiation exposure. However, the deletion also exacerbates bone loss, potentially due to reduced secretion of key bone-regulating factors such as BMP-2 and TGFβ. This highlights a complex interplay between endothelial TLR9 signaling, vascular damage, brain function, and skeletal integrity, emphasizing the need for a balanced approach when targeting TLR9 in radiation.