A new dynamic in vitro modular capillaries-venules modular system: cerebrovascular physiology in a box

Abstract

Background: The study of the cerebrovascular physiology is crucial to understand the pathogenesis of neurological disease and the pharmacokinetic of drugs. Appropriate models in vitro often fail to represent in vivo physiology. To address these issues we propose the use of a novel artificial vascular system that closely mimics capillary and venous segments of human cerebrovasculature while also allowing for an extensive control of the experimental variables and their manipulation.

Results: Using hollow fiber technology, we modified an existing dynamic artificial model of the blood-brain barrier (BBB) (DIV-capillary) to encompass the distal post-capillary (DIV-venules) segments of the brain circulatory system. This artificial brain vascular system is comprised of a BBB module serially connected to a venule segment. A pump generates a pulsatile flow with arterial pressure feeding the system. The perfusate of the capillary module achieves levels of shear stress, pressure, and flow rate comparable to what observed in situ. Endothelial cell exposure to flow and abluminal astrocytic stimuli allowed for the formation of a highly selective capillary BBB with a trans-endothelial electrical resistance (TEER; >700 ohm cm2) and sucrose permeability (< 1X10-u cm/sec) comparable to in vivo. The venule module, which attempted to reproduce features of the hemodynamic microenvironment of venules, was perfused by media resulting in shear stress and intraluminal pressure levels lower than those found in capillaries. Because of altered cellular and hemodynamic factors, venule segments present a less stringent vascular bed (TEER <250 Ohm cm2; Psucrose > 1X10-4 cm/sec) than that of the BBB. Abluminal human brain vascular smooth muscle cells were used to reproduce the venular abluminal cell composition.

Conclusion: The unique characteristics afforded by the DIV-BBB in combination with a venule segment will realistically expand our ability to dissect and study the physiological and functional behavior of distinct segments of the human cerebrovascular network.

Figure 1

Schematic outline of the DIV capillary-venules model. Note how the system recapitulates both rheological and cellular characteristics of the corresponding in vivo cerebrovascular segments.

Figure 2

Rheological characteristics of the DIV capillary-venule system. Panel A: Hemodynamic profile of capillary and venule segments in respect to perfusion. Panel B: Note that the presence of inter module connector between the capillary and venule lumens did not alter the rheological profile of flow. The asterisk “*” indicates a statistically significant difference in transmural pressure between capillary and venules (n=4; p<0.05).

Figure 3

Side by side comparison between in vitro capillary and venule vascular beds. Panel A: Note how the capillary system allows for the formation of a very stringent vascular bed (high TEER) in comparison to venules (low TEER). (Panel B). Note also that capillary segments established under venules level of shear stress and venules module exposed to capillary shear stress levels formed a comparable low stringent barrier suggesting that both abluminal astrocytes and high shear stress levels are necessary to develop a tight vascular bed (Panel C) TEER and sucrose permeability correlation in capillaries and venules modules. The sigmoid curve symbolize the ideal correlation between TEER and permeability previously determined by us [55]. Note the difference of ≈ 2 order of magnitude between capillaries (less permeable) and venules (most permeable). The more stringent vascular bed formed in the capillary module can discriminate drug permeability based on the octanol-water partition coefficient (XlogP) with a significantly higher degree of selectivity than venules (Panel D). The asterisk “*” indicates a statistically significant difference (n=4; p<0.05).

Figure 4

Functional characterization of the DIV capillary-venule system. Hyperosmolar opening of the BBB in DIV models was assessed by real time measurements of TEER. Similar to what observed in vivo the differential magnitude and the transient nature of the vascular opening is indicative of the formation of capillary and venule vascular beds that closely mimic the physiological response of the corresponding cerebrovascular segments in situ.

Figure 5

Differential bioenergetic behavior of in vitro capillary and venule vascular systems. Panel A: Glucose consumed versus lactate produced over time in capillary and venule modules. Note the significant difference between the glucose consumption and lactate production ratio (R) in the capillary module (R≅1) and in the venule (R≅1.5). This indicates that in contrast to venule segments, the brain capillary cellular elements have increased propensity towards the use of aerobic-based glucose metabolism. The asterisk “*” indicates a statistically significant difference (n=4; p<0.05) versus parallel systems established under static conditions.