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Regulation of blood flow in the microcirculation


Anh Thuc Ngo


In the microcirculation blood flow is highly regulated and dependent on the metabolic activityof the tissues. Mechanisms involved in the coupling of changes in tissue oxygen tension due tochanges in the metabolic activity of the tissue play an important role. In the systemic arterysystem hypoxia causes dilatation of arteries and arterioles (hypoxic vasodilatation), whereashyperoxia causes constriction (hyperoxic vasoconstriction).The aim of this PhD study is to examine: I) mechanisms involved in hypoxic vasodilatation andhyperoxic vasoconstriction, and II) whether localized hypoxia or hyperoxia is able to induce aconducted vasomotor response in mouse cremaster arterioles in situ.In anesthetized C57BL/6J male mice, cremaster arterioles (~30 μm) where examined duringsuperfusion with a Krebs’ buffer equilibrated with gas mixtures containing 0% (low oxygensuperfusate) or 21% O2 (high oxygen superfusate), 5% CO2 and N2 balance. Low oxygensuperfusates (PO2 ~15 mmHg) caused dilatation of the arterioles and high oxygen superfusates(PO2 ~160 mmHg) caused vasoconstriction. The role of ATP-sensitive K+ channels (KATPchannels) was tested. Application of 10 μM glibenclamide (inhibitor of KATP channels) in thesuperfusate abolished both vasodilatation and constriction to low and high oxygen tension,indicating that KATP channels are involved in both hypoxic vasodilatation and hyperoxicvasoconstriction. Red blood cells (RBCs) have been proposed to release ATP and/or NO inresponse to hypoxia, which acts on the vascular wall causing vasodilatation. In cremasterarterioles devoid of RBCs, achieved by buffer perfusion via a cannula in the abdominal aorta ofthe animals, the cremaster arterioles showed the same degree of dilatation and constriction tolow and high oxygen as in the intact blood-perfused arteriole. This indicates that RBCs are notessential for hypoxic vasodilatation. In addition several potential pathways were evaluated.Application of DPCPX (inhibitor of adenosine A1 and A2 receptors) and L-NAME (inhibitor ofNO-synthase) did not affect vasomotor responses to low or high oxygen superfusate. Thisindicates that adenosine and NO release are not involved in the regulation of blood flow induced by changes in tissue oxygen tension. The roles of prostaglandins and 20-HETE were also tested.While application of indomethacin (inhibitor of cyclooxygenase) in the superfusate caused atemporary inhibition of vasodilatation during low oxygen superfusate, the arterioles quicklyregained their response to low and high oxygen superfusates. This suggests that prostaglandinsare not involved in hypoxic vasodilatation in mouse cremaster arterioles. Conversely,application of DDMS (inhibitor of 20-HETE production) inhibited vasoconstriction to highoxygen superfusates, indicating that 20-HETE is involved in hyperoxic vasoconstriction.Using a custom-built gas exchange chamber with a small aperture covered by a gas-permeablemembrane, we were able to induce highly localized oxygen tension changes to parts of thecremaster microcirculation independent on the oxygen tension in the blood vessels supplying thetissue. We showed that induction of localized low oxygen and high oxygen caused a localvasodilatation and constriction, which was conducted along the arteriole to a site, located 1000μm upstream (distant site). Functional transmural damage to a short segment of the arteriolelocated midway (500 μm) between the local site and distant site, utilizing a method called “lightdye treatment”, abolished the conducted response, but not the local response. This indicates thatchanges in oxygen tension are able to induce a conducted vasomotor response.In a separate study we examined if low or high oxygen tension caused any change in theintracellular Ca2+ concentration of isolated mouse cremaster arterioles using fluorescentmicroscopy. Cremaster arterioles were isolated by gentle microdissection, loaded with Fura-PE3(calcium fluorescent dye) and placed in a small chamber which was perfused with low or highoxygen Krebs’ buffer. While there was a significant increase in the intracellular Ca2+concentration during application of 75 mM KCl or 100 μM phenylephrine, no change in theintracellular Ca2+ concentration was seen during change from low to high oxygen perfusate.Possible explanations for these findings could be: a) that the oxygen sensing mechanism wasdamaged during the dissection procedure, b) changes in oxygen tension does not cause changesin the intracellular Ca2+ concentration, but rather changes the Ca2+ sensitivity of the contractiveapparatus and c) that the arterioles were not perfused and pressurized, which rendered themunresponsive to changes in oxygen tension. To examine the last possibility, mouse mesentericarteries (diameter ~200 μm) were isolated and mounted in a pressure myography. Duringintraluminal pressure of 40 mmHg, change from low to high oxygen caused no change in vesseldiameter, however when intraluminal pressure was raised to 80-120 mmHg, change in theoxygen tension caused change in vessel diameter. This indicates that the vessels need to bepressurized in order to be able to sense and react to changes in the oxygen tension.