Brain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier

Abstract

Endothelial glycocalyx coats healthy vascular endothelium and plays an important role in vascular homeostasis. Although cerebral capillaries are categorized as continuous, as are those in the heart and lung, they likely have specific features related to their function in the blood brain barrier. To test that idea, brains, hearts and lungs from C57BL6 mice were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx, and examined using scanning and transmission electron microscopy. We found that endothelial glycocalyx is present over the entire luminal surface of cerebral capillaries. The percent area physically covered by glycocalyx within the lumen of cerebral capillaries was 40.1 ± 4.5%, which is significantly more than in cardiac and pulmonary capillaries (15.1 ± 3.7% and 3.7 ± 0.3%, respectively). Upon lipopolysaccharide-induced vascular injury, the endothelial glycocalyx was reduced within cerebral capillaries, but substantial amounts remained. By contrast, cardiac and pulmonary capillaries became nearly devoid of glycocalyx. These findings suggest the denser structure of glycocalyx in the brain is associated with endothelial protection and may be an important component of the blood brain barrier.

Figure 1

Scanning electron micrographs showing the ultrastructure of continuous capillaries in the (A) brain, (B) heart and (C) lung. Upper panels: without lanthanum nitrate staining. Lower panels: with lanthanum nitrate staining for visualization of endothelial glycocalyx. Panels on the right are expanded views of those on the left. Continuous capillaries have a continuous basement membrane, and the endothelial glycocalyx can be seen on the surface of the vascular endothelial cells.

Figure 2

Transmission electron microscopic analysis of continuous capillaries. (A–C) Cerebral (A), cardiac (B) and pulmonary (C) capillaries with lanthanum nitrate staining. The endothelial glycocalyx covers the surface of vascular endothelial cells. (D) Percent area covered by endothelial glycocalyx in capillaries of brain, heart and lung. Bars indicate means ± SE. *p < 0.05 vs brain.

Figure 3

Characterization of LPS-injected mice. (A) Survival after LPS administration. Forty-eight h after LPS administration, the survival rate of mice was 22% (11/50 injected mice). (B) Plasma syndecan-1 concentrations at the indicated times after LPS administration. *p < 0.05 vs. before LPS administration. (C) Extravasated Evans blue levels in brain, heart and lung at the indicated times after LPS administration. Levels were normalized to organ weight. Bars indicate means ± SE. *p < 0.05 vs before LPS administration in each organ.

Figure 4

Scanning electron micrographs showing the ultrastructure of continuous capillaries 48 h after LPS administration in the (A) brain, (B) heart and (C) lung. Upper panels: without lanthanum nitrate staining, Lower panels: with lanthanum nitrate staining for visualization of endothelial glycocalyx. Right panels are expanded views of the left panels in each figure. After LPS administration, the endothelial glycocalyx is degraded on the surface of the vascular endothelium in the heart and lung, but the glycocalyx is maintained in the brain.

Figure 5

Transmission electron microscopic analysis of continuous capillaries 48 h after LPS administration. (A–C) Cerebral (A), cardiac (B) and pulmonary (C) capillaries with lanthanum nitrate staining. The endothelial glycocalyx has degraded on the surface of vascular endothelial cells in the heart and lung, but not in the brain. (D) Percent area covered by endothelial glycocalyx in capillaries of brain, heart and lung. Bars indicate means ± SE. *p < 0.05 vs brain.