Automated image analysis for diameters and branching points of cerebral penetrating arteries and veins captured with two-photon microscopy.

The present study was aimed to characterize 3-dimensional (3D) morphology of the cortical microvasculature (e.g., penetrating artery and emerging vein), using two-photon microscopy and automated analysis for their cross-sectional diameters and branching positions in the mouse cortex. We observed that both artery and vein had variable cross-sectional diameters across cortical depths. The mean diameter was similar for both artery (17 ± 5 µm) and vein (15 ± 5 µm), and there were no detectable differences over depths of 50-400 µm. On the other hand, the number of branches was slightly increased up to 400-µm depth for both the artery and vein. The mean number of branches per 0.1 mm vessel length was 1.7 ± 1.2 and 3.8 ± 1.6 for the artery and vein, respectively. This method allows for quantification of the large volume data of microvascular images captured with two-photon microscopy. This will contribute to the morphometric analysis of the cortical microvasculature in functioning brains.

3D analysis of intracortical microvasculature during chronic hypoxia in mouse brains.

The purpose of this study is to determine when and where the brain microvasculature changes its network in response to chronic hypoxia. To identify the hypoxia-induced structural adaptation, we longitudinally imaged cortical microvasculature at the same location within a mouse somatosensory cortex with two-photon microscopy repeatedly for up to 1 month during continuous exposure to hypoxia (either 8 or 10% oxygen conditions). The two-photon microscopy approach made it possible to track a 3D pathway from a cortical surface arteriole to a venule up to a depth of 0.8 mm from the cortical surface. The network pathway was then divided into individual vessel segments at the branches, and their diameters and lengths were measured. We observed 3-11 vessel segments between the penetrating arteriole and the emerging vein over the depths of 20-460 µm within the 3D reconstructed image (0.46 × 0.46 × 0.80 mm(3)). The average length of the individual capillaries (<7 µm in diameter) was 67 ± 46 µm, which was not influenced by hypoxia. In contrast, 1.4 ± 0.3 and 1.2 ± 0.2 fold increases of the capillary diameter were observed 1 week after exposure to 8 % and 10% hypoxia, respectively. At 3 weeks from the exposure, the capillary diameter reached 8.5 ± 1.9 and 6.7 ± 1.8 µm in 8% and 10 % hypoxic conditions, respectively, which accounted for the 1.8 ± 0.5 and 1.4 ± 0.3 fold increases relative to those of the prehypoxic condition. The vasodilation of penetrating arterioles (1.4 ± 0.2 and 1.2 ± 0.2 fold increases) and emerging veins (1.3 ± 0.2 and 1.3 ± 0.2 fold increases) showed relatively small diameter changes compared with the parenchymal capillaries. These findings indicate that parenchymal capillaries are the major site responding to the oxygen environment during chronic hypoxia.