Changes in cortical microvasculature during misery perfusion measured by two-photon laser scanning microscopy

Abstract

This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1, 7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9%±3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5%±19.7%, 57.2%±32.3%, and 32.0%±10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9%±1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.

Figure 1

Two-photon laser scanning microscopy images of a pial arteriole before and after unilateral common carotid artery occlusion (UCCAO) during both normocapnia and hypercapnia taken at the cerebral cortical surface. Scale bar, 10 μm. After UCCAO, red blood cell speed seems to have dramatically decreased. Autoregulatory vasodilation was calculated as the percentage change in diameter from the preoperative value on each experimental day. Vascular response to hypercapnia was calculated as the percentage change in diameter during hypercapnia in reference to normocapnia. Solid lines perpendicular to the vessels indicate the diameter of the arterioles in each condition. Yellow dashed lines indicate the position of the vessel wall during normocapnia before UCCAO (a). Green dashed lines indicate the position of the vessel wall during normocapnia after UCCAO (c). (a–d) Indicate the vessel diameter during normocapnia before UCCAO, during hypercapnia before UCCAO, during normocapnia after UCCAO, and during hypercapnia after UCCAO, respectively.

Figure 2

Vascular responses to hypercapnia during chronic hypoperfusion in the bilateral cerebral hemisphere. (A, B) Time–response curves for the normalized increase in cerebral blood flow (CBF) response to hypercapnia during chronic hypoperfusion in the hemisphere ipsilateral to unilateral common carotid artery occlusion (UCCAO) (A) and in the hemisphere contralateral to UCCAO (B). The horizontal bars indicate the 5% CO₂ inhalation period. These data were normalized to the pre-CO₂ inhalation level (20 seconds before 5% CO₂ inhalation). Each response curve represents the mean for all animals on each measurement day. The error bars indicate standard deviation. (C) Mean percentage increase in CBF in the bilateral hemisphere within 10 to 20 seconds of 5% CO₂ inhalation. Ipsilateral hemisphere (Ipsi, black); contralateral hemisphere (Contra, white). The error bars indicate standard deviation. *P<0.05, **P<0.01.

Figure 3

(A) Longitudinal imaging of cortical vessels at the cortical surface in the hemisphere ipsilateral to unilateral common carotid artery occlusion (UCCAO). The arterioles are dilated significantly after UCCAO. Before UCCAO, the arterioles were dilated during hypercapnia. The dilation observed during hypercapnia was unnoticeable after UCCAO. Scale bar, 50 μm. (B) Longitudinal diameters in the cortical microvessels during normocapnia and hypercapnia in the hemisphere ipsilateral to UCCAO. (C) Longitudinal percentage changes in cortical microvessel diameter from the preoperative value. These findings represent the diameter response to decreased cerebral perfusion pressure (CPP; i.e., autoregulatory vasodilation). The microvessels dilated progressively and reached a maximum diameter at 14 days after UCCAO. (D) Longitudinal cortical microvessel diameter responses to hypercapnia. The diameter responses to hypercapnia in the arterioles were significantly decreased after UCCAO. The error bars indicate standard deviation. A, arteriole; C, capillary; V, venule. Normocapnia (white); hypercapnia (black). *P<0.05, **P<0.01 (against preoperative value).

Figure 4

(A) Longitudinal imaging of cortical vessels at the cortical surface in the hemisphere contralateral to unilateral common carotid artery occlusion (UCCAO). Scale bar, 50 μm. (B) Longitudinal diameters in cortical microvessels during normocapnia and hypercapnia in the hemisphere contralateral to UCCAO. (C) Longitudinal percentage changes in cortical microvessel diameter from the preoperative value in the hemisphere contralateral to UCCAO. Although arterioles were slightly dilated 7 days after UCCAO, statistically significant dilation was not found in any of the three microvasculature components throughout the experimental period. (D) Longitudinal vascular responses to hypercapnia in cortical microvessels in the hemisphere contralateral to UCCAO. There were no significant differences in responses throughout the experimental period. The error bars indicate standard deviation. A, arteriole; V, venule. Normocapnia (white); hypercapnia (black).

Figure 5

Correlation between the diameter responses to decreased cerebral perfusion pressure (CPP) and those to hypercapnia in the arterioles for the hemisphere ipsilateral to unilateral common carotid artery occlusion (UCCAO) in all mice during the entire experimental period. There was a significant negative correlation (R²=0.806; P<0.0001).

Figure 6

Magnetic resonance imaging experiments obtained 30 days after unilateral common carotid artery occlusion. The panel on the left shows a T2-weighted image. The panel on the right shows a diffusion-weighted image. The images were acquired at the area including the parietal cortex (PC) and hippocampus (HC). No abnormal changes in signal intensity were observed. The white arrow indicates the cover glass.