3D surface analysis of hippocampal microvasculature in the irradiated brain

Cranial irradiation used to control CNS malignancies can also disrupt the vasculature and impair neurotransmission and cognition. Here we describe two distinct methodologies for quantifying early and late radiation injury in CNS microvasculature. Intravascular fluorescently labeled lectin was used to visualize microvessels in the brain of the irradiated mouse 2 days post exposure and RECA-1 immunostaining was similarly used to visualize microvessels in the brain of the irradiated rat 1-month post exposure. Confocal microscopy, image deconvolution and 3-dimensional rendering methods were used to define vascular structure in a ∼4 × 10(7) μm(3) defined region of the brain. Quantitative analysis of these 3D images revealed that irradiation caused significant short- and long-term reductions in capillary density, diameter and volume. In mice, irradiation reduced mean vessel volume from 2,250 to 1,470 μm(3) and mean vessel diameter from 5.0 to 4.5 μm, resulting in significant reductions of 34% and 10%, in the hippocampus respectively. The number of vessel branch points and area was also found to also drop significantly in mice 2 days after irradiation. For rats, immunostaining revealed a significant, three-fold drop in capillary density 1 month after exposure compared to controls. Such radiation-induced disruption of the CNS microvasculature may be contributory if not causal to any number of neurocognitive side effects that manifest in cancer patients following cranial radiotherapy. This study demonstrates the utility of two distinct methodologies for quantifying these important adverse effects of radiotherapy. Environ. Mol. Mutagen. 57:341-349, 2016. © 2016 Wiley Periodicals, Inc.