David N. Damon

Central Role of Connexin40 in the Propagation of Electrically Activated Vasodilation in Mouse Cremasteric Arterioles In Vivo

&NA; When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40‐/‐) mice to study vasomotor responses induced by 10‐second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 &mgr;m upstream). In wild‐type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40‐/‐ mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 &mgr;mol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40‐/‐ mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40‐/‐ mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40‐/‐ mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40‐/‐ animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40‐dependent vasodilator response that spreads along the vessel length without decay. (Circ Res. 2003;92:793–800.)

Augmented tissue oxygen supply during striated muscle contraction in the hamster. Relative contributions of capillary recruitment, functional dilation, and reduced tissue PO2.

To investigate the relative contributions of alterations in blood flow, capillary density, and tissue PO2 to elevated oxygen delivery in working muscle, we conducted experiments on the suffused hamster cremaster muscle, using in vivo microscopic techniques. Muscle PO2 was measured during striated muscle twitch contraction at 1 Hz. Tissue oxygenation was changed by using suffusion solutions equilibrated with 0%, 5%, 10%, 21%, or 50% oxygen. Contraction caused an increase in capillary density (capillary recruitment), whose magnitude was related to the equilibration gas and, thus, to the suffusate PO2. Capillary recruitment first increased as the oxygen content was raised, peaked with 10% oxygen, and then diminished with higher oxygen content. Arteriolar functional dilation was also observed; when oxygen was raised above 21%, dilation was decreased. The data suggest that oxygen supply is increased primarily by arteriolar conductance changes with low suffusion solution oxygen (0% to 5%), and by capillary recruitment and increased PO2 gradients above 10% oxygen. When vasomotor tone was increased by addition of norepinephrine to the suffusion medium, the changes observed were similar to those observed when oxygen was increased. Therefore, we propose that the altered microvascular responses during vasoconstriction are a function of vascular tone rather than the levels of tissue PO2. A model is proposed which may partially explain the relations among vascular tone, functional dilation, and capillary recruitment. Our data also suggest that tissue PO2 may not be precisely regulated about a narrowly defined set point in this striated muscle but that, instead, tissue PO2 is a dependent variable controlled by the integrated effects of capillary recruitment, functional vasodilation, and altered metabolism.

Quantitative morphology of arterioles from the hamster cheek pouch related to mechanical analysis

Abstract The structure of 20 second-order arterioles from the hamster cheek pouch was quantitated after histological fixation at physiological pressures. Dilated and contracted vessels with respective internal diameters of 66 ± 9.2 and 30 ± 9.1 μm (SD) were studied. Electron microscopy of four dialted arterioles revealed a combined intima-media thickness of 3.2 ± 1.5 μm with the following composition determined by stereological point counting: endothelial cells (20%), endothelial basement membrane (3%), a continuous elastic lamina (8%), smooth muscle cells (49%), and medial extracellular space (20%). Light microscopy of whole mounts of cheek pouches and isolated arterioles stained with hematoxylin was used to measure orientation and lengths of both smooth muscle and endothelial cell nuclei. The smooth muscle orientation was not significantly different in dilated or contracted arterioles. For all of the muscle orientation measurements combined, the mean pitch angle was 1.1 ± 5.9° ( N = 1000) with respect to the circumferential axis of the vessel. Comparison of dilated and contracted arterioles indicated a significant decrease in the muscle cell and nuclear length, but no detectable change in either orientation or nuclear length of the longitudinally oriented endothelial cells. Wall areas measured from transverse sections of dilated and contracted vessels did not change upon contraction. Stereological estimates of relaxed smooth muscle cell length averaged approximately 60 μm. The manner in which structural parameters affect arteriolar mechanical properties is discussed, and it is concluded that, in hamster pouch arterioles, over 99% of the developed smooth muscle force is transmitted circumferentially.

Inaccuracies in blood flow estimates in microvessels during arteriolar vasoconstriction

The effect of vasomotor tone on blood flow estimates was evaluated in the hamster cheek pouch and cremaster muscle microcirculation. The products of arteriolar cross-sectional area and red blood cell velocity were calculated in three different cases: (1) at arteriolar bifurcations, (2) in short segments of an arteriole constricted by iontophoretic application of norepinephrine, and (3) at randomly selected second- and third-order arterioles. Vasodilation of the microcirculation was induced by topical application of adenosine. Vasoconstriction was induced by elevation of superfusion solution PO2. If true volume flow is accurately estimated by this method then: the sum of measured branch flows at a bifurcation should equal feed flow; measured flow through constricted arteriolar segments should equal flow proximal or distal to the constricted segment; and, following experimental manipulations, relative changes in estimated flow in second- and third-order arterioles should be equal. Our findings suggest that the blood flow estimates were not always accurate. The sum of branch flows was equal to feed flow only across bifurcations with low or resting vascular smooth muscle tone. During vasoconstriction, feed flow averaged 40% higher than the sum of downstream flows. In addition, estimated flow was 15% lower in constricted segments of an arteriole compared to dilated contiguous segments of the vessel. During alterations in vasomotor state, estimated fractional changes in flow in second- and third-order arterioles differed by more than sixfold. Therefore, blood flow estimates with the dual-slit method may not be reliable under conditions of high vasomotor tone. We speculate that the error may result largely from uncertainties in the diameter measurement.

Venular reactivity in the hamster cheek pouch and cremaster muscle

Studies of local blood flow control usually ignore vasomotor activity of the venous side of the microcirculation, but there is evidence that venules from a variety of tissues respond to pharmacological (Altura, 1978, 1981), and physiological stimuli (Marshall and Tandon, 1984). To determine whether venous reactivity may be involved in the control of tissue oxygen delivery, we examined the responsiveness of venules of the hamster cheek pouch and cremaster muscle to changes in super-fusion solution oxygen (0,) content. Responses were simultaneously obtained from arterioles for comparison with venous responsiveness and to estimate overaIl microvascular reactivity. As an independent estimate of vascular reactivity, the response to norepinephrine (NE) was also assessed.