Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.     

Hemodynamics and vascular sensitivity to circulating norepinephrine in normal skin and delayed and acute random skin flaps in the pig

Cutaneous circulation in 4 X 10 cm skin samples and delayed and acute random skin flaps constructed on the flanks of castrated Yorkshire pigs (13.3 +/- 0.7 kg; n = 12) were studied during intravenous infusion (0.5 ml per minute) of 5% dextrose solution (vehicle) and 5% dextrose containing norepinephrine (1 microgram/kg per minute). Total and capillary blood flow and A-V shunt flow were measured by the radioactive microsphere technique 6 hours after the raising of 4 X 10 cm single-pedicle acute and delayed random skin flaps using the technique and calculations published previously. Fluorescein dye test was also performed to assess vascular perfusion. It was observed that the capillary blood flow in the single-pedicle delayed skin flaps was similar to that in the normal skin, and the maintenance of this normal skin blood flow was not due to the closing of A-V shunt flow in the delayed skin flaps. Similarly, the significant (p less than 0.01) decrease in capillary blood flow and distal perfusion in the acute skin flaps compared with the delayed skin flaps was not due to the opening of A-V shunts in the acute skin flaps. There was no evidence to indicate that A-V shunt flow per se was the primary factor for the regulation of capillary blood flow in the acute and delayed skin flaps in the pig. Our data seemed to indicate that tissue ischemia in the distal portion of acute skin flaps was likely the result of vasoconstriction of the small random arteries which supplied blood to arterioles and A-V shunts, and locally released neurohumoral substances may play an important role in the pathogenesis of vascular resistance and ischemia in the acute skin flaps.     

Myocutaneous flap ischemia: flow dynamics following venous and arterial obstruction.

To further clarify the pathogenesis of the poorer prognosis in skin flaps exposed to venous stasis compared with arterial insufficiency, a microsphere study was conducted in bilateral rectus abdominis island flaps in seven pigs. The relationship between capillary blood flow and arteriovenous (A-V) shunting was studied during progressive 1-hour intervals of arterial insufficiency and venous stasis and during 3 hours of reperfusion. Under controlled conditions, total blood flow was reduced from 100 percent to both 50 and 25 percent by application of an adjustable clamp on the artery supplying one flap and on the vein draining the contralateral flap. The relative distribution between A-V shunt flow and capillary blood flow was different in arterial insufficiency when compared with venous stasis at both the 50 percent and the 25 percent blood flow levels. In the arterial insufficiency flaps, the A-V shunt flow and capillary blood flow shared the total blood flow in the following percentages: 64/36 (at 100 percent total blood flow), 44/56 (at 50 percent total blood flow level), and 22/78 (at 25 percent total blood flow level). In the venous stasis flaps, the A-V shunt flow and the capillary blood flow shared the total blood flow in percentages of 70/30, 66/34, and 55/45, respectively. Hence, in arterial insufficiency flaps, capillary blood flow was spared by a relatively greater decline in A-V shunting compared with venous stasis flaps. Redistribution of capillary blood flow from subcutaneous tissue to muscle was observed, whereas blood flow was equally distributed throughout the length of the flaps at all flow levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascularization of plexus myentericus (Auerbach) in the large intestine of the cat

1. Coincidental preparation of the intramuscular vascular bed and the plexus myentericus (Auerbach) of the cat's large intestine by India-ink method and silverimpregnation allowed to demonstrate independent vascularisation of ganglia and nerve-branches of the plexus Auerbach. 2. Each ganglion is surrounded by a capillary network widely independently existing of the intramuscular capillary bed. The preferred innervated terminal arterioles and especially the sphincteric capillaries opening into the periganglionic capillary network and the numerous arterio-venous short-circuits in its marginal area suggest to conclude a differentiated regulation of blood supply.     

Effects of somatostatin on intestinal circulation and oxygen consumption

The effects of intraarterial administration of somatostatin upon intestinal blood flow, intestinal capillary surface area, oxygen consumption and intestinal motor activity were measured in anesthetized dogs. Blood flow to the segment of distal ileum was measured with an electromagnetic blood flow meter, and arteriovenous oxygen difference (AVO2) was determined spectrophotometrically. Intestinal oxygen consumption was calculated as the product of AVO2 and total blood flow. The clearance of 86Rb was measured to estimate the density of the perfused intestinal capillaries. Changes in blood flow distribution were estimated from the distribution of radiolabelled microspheres. Intestinal motor activity was monitored from changes in intraluminal pressure. Somatostatin induced a dose-related decrease in intestinal blood flow, capillary surface area and intestinal oxygen consumption. A significant increase in intestinal motor activity was also observed. The data of this study indicate that somatostatin acts on smooth muscle of both arterioles and precapillary sphincters and results in a potent vasoconstriction in the intestinal microcirculation.     

Pulmonary vascular K+ channel expression and vasoreactivity in a model of congenital heart disease

K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K Ca ) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K Ca channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K Ca channel expression is increased and TASK is decreased.     

Functional Organization of the Teleost Gill

The circulation of the gills has been studied in the perch, trout and eel combining the conventional histological methods and casting techniques. The existence of two blood pathways in each gill arch was confirmed. 1 — An arterio-arterial pathway assuming the respiratory function. It includes the afferent branchial artery and in each primary lamella the afferent primary artery, the secondary lamellae capillaries and the primary and branchial efferent arteries. 2 — An arterio-venous pathway arising from both the branchial artery, in the gill arch, and the primary arteries in each primary lamella. This pathway includes the central venous sinus of the primary lamella, several small veins and is finally connected with the branchial veins. 3 — The lack of connections between afferent primary arteries and cvs in the trout and the perch makes impossible a direct blood flow from the afferent to the efferent artery (shunt). In the eel connections between cvs and both afferent and efferent arteries do not mean that a shunt is operating according to the pressure gradient.     

The flow of sickle-cell blood in the capillaries.

Oxygen tension levels and red cell velocities for the flow of sickle-cell blood in the capillaries are determined by using the Krogh model for oxygen transport and lubrication theory for the cell motion. The coupling and interaction between these arises from the red cell compliance, which is assumed to vary with the oxygen concentration. Microsieving data is used to establish an upper bound for this relationship. Calculations are carried out for a range of capillary sizes, taking into account the rightward shift of the oxyhemoglobin dissociation curve and the reduced hematocrit of sickle-cell blood, and are compared to, as a base case, the flow of normal blood under normal pressure gradient. The results indicate that under normal pressure gradients the oxygen tensions and cell velocities for sickle blood are considerably higher than for normal blood, thus acting against the tendency for cells to sickle, or significantly change their rheological properties, in the capillaries. Under reduced pressure gradients, however, the concentrations and velocities drop dramatically, adding to the likelihood of such shape or flow property changes.     

The distribution of ventilation-perfusion ratios in the lungs of newborn foals

The distributions of ventilation-perfusion ratios, and the effects of 100% oxygen administration on the distributions, were studied in 3 foals from 4h to 9 days of age, using the multiple inert gas elimination technique. The distributions were calculated from the pulmonary clearance of 6 inert gases following infusion into a peripheral vein of a solution containing the inert gases. The results from a total of 8 studies showed several consistent features. The major findings were (i) the absence of low ventilation-perfusion ratios, i.e. regions where blood flow was greatly in excess of ventilation; (ii) the presence of variable right to left shunt; (iii) a reduction in this shunt with increasing post-natal age; (iv) the presence of a separate high mode of ventilation-perfusion ratios where ventilation was greatly in excess of blood flow and; (v) the observation that breathing enriched oxygen mixtures for 40 min did not increase the right to left shunt in any foal at any age studied. These studies indicate that hypoxaemia in the neonatal foal is attributable to right to left shunt which may be intrapulmonary or intracardiac, or both, rather than overperfusion of poorly ventilated lungs.     

Strength of hypoxic vasoconstriction determines shunt fraction in dogs with atelectasis

We investigated the degree to which strength of pulmonary hypoxic vasoconstriction affects perfusion of pulmonary shunt pathways in acute atelectasis. In 17 intact supine dogs (anesthetized, paralyzed, and ventilated) we produced left lower lobe atelectasis by occluding the lobar bronchus during oxygen inhalation. Subsequently, shunt fraction (reflecting perfusion of that lobe) was measured using an SF6 infusion while the dogs breathed room air; the mean was 26% (range 14-40%). Pulmonary pressor response to hypoxia was assessed in 13 dogs using the increase in pulmonary end-diastolic gradient (PDG) produced by inhalation of 10% oxygen. Those animals with the largest increase in pulmonary diastolic gradient had the smallest shunt fraction while breathing room air, whereas those with the smallest response had the largest shunt fraction. The contribution of local hypoxia to vasoconstriction in the shunt pathway was assessed in 13 dogs breathing room air by measuring the increase in shunt fraction produced by infusing prostaglandin E1 (PGE1). Those with the largest increase in shunt fraction had the smallest pre-PGE1 shunt fraction. Thus the strength of pulmonary vascular reactivity to hypoxia markedly influences the degree of vasoconstriction in shunt pathways and is a major determinant of shunt pathway perfusion.     

A computational study of the effect of capillary network anastomoses and tortuosity on oxygen transport

The objective of this study was to investigate the effects of capillary network anastomoses and tortuosity on oxygen transport in skeletal muscle, as well as the importance of muscle fibers in determining the arrangement of parallel capillaries. Countercurrent flow and random capillary blockage (e.g. by white blood cells) were also studied. A general computational model was constructed to simulate oxygen transport from a network of blood vessels within a rectangular volume of tissue. A geometric model of the capillary network structure, based on hexagonally packed muscle fibers, was constructed to produce networks of straight unbranched capillaries, capillaries with anastomoses, and capillaries with tortuosity, in order to examine the effects of these geometric properties. Quantities examined included the tissue oxygen tension and the capillary oxyhemoglobin saturation. The computational model included a two-phase simulation of blood flow. Appropriate parameters were chosen for working hamster cheek-pouch retractor muscle. Our calculations showed that the muscle-fiber geometry was important in reducing oxygen transport heterogeneity, as was countercurrent flow. Tortuosity was found to increase tissue oxygenation, especially when combined with anastomoses. In the absence of tortuosity, anastomoses had little effect on oxygen transport under normal conditions, but significantly improved transport when vessel blockages were present.     

Characteristic features of microvascular responses of healthy humans to simulated emotional stress

Characteristic features of microvascular responses of healthy humans to simulated emotional stress (ES) were studied using computer-aided laser Doppler flowmetry. It was shown that, in ES, responses of individual regions of the microvasculature are relatively autonomous and different. In the nail bed, where the arteriolar and the arteriolovenular shunt tone depends on the neurogenic sympathoadrenal regulation, the responses are more pronounced than in the lower arm microvessels. The rearrangements of the nail bed and lower arm microvasculature in response to a simulated situation of ES are different, which is likely to be determined by both their different vascular architecture and other factors (the pressure gradient, metabolic requirements of tissues, etc.). It was established that the ES level determines an increase in the contributions of the neurogenic, myogenic, respiratory, and cardiac components to the modulation of the intensity of microcirculation, increasing the shunt value and the blood flow in microvessels.     

Metabolic models of microcirculatory regulation.

The functions and integrity of body tissues are critically dependent on an adequate oxygen supply. Because the transport of oxygen to the cells is intimately linked to the microcirculation, the concept of microcirculation-metabolism coupling has received much attention. In essence, the metabolic theory of intrinsic control of the microcirculation states that microvascular tone is locally modulated to maintain adequate oxygen levels in the parenchymal cells. We propose a two-component control system for the regulation of tissue O2 delivery in accordance with metabolic needs. A precapillary sphincter control mechanism maintains tissue PO2 by governing the number of perfused capillaries. Functional capillary density in turn determines surface area available for diffusion and capillary-to-cell diffusion distance. On the other hand, the arteriolar control system modulates local blood flow in accordance with parenchymal O2 utilization and thereby minimizes changes in capillary PO2 when the O2 availability/demand ratio is decreased. We propose that the precapillary sphincters are more sensitive to changes in tissue PO2 than are the flow-regulating arterioles. Consequently, for mild stresses, adequate tissue oxygenation is maintained mainly by precapillary sphincter control of diffusion parameters without the need for changes in blood flow. However, as metabolic stresses become greater, blood flow regulation becomes the dominant factor in the control of tissue O2 delivery. Thus, by working in concert, the local mechanisms regulating microvascular resistance and effective capillary density provide a wide margin of safety against the development of cellular hypoxia.     

Blood Vessels in the Hind Limb of the Mallard (Anas platyrhynchos): Anatomical Evidence for a Sphincteric Action of Shunt Vessels in Connection with the Arterio-venous Heat Exchange System

Midtgård, U. 1980. Blood vessels in the hind limb of the Mallard (Anas platyrhynchos): anatomical evidence for a sphincteric action of shunt vessels in connection with the arterio-venous heat exchange system. (Institute of Comparative Anatomy, University of Copenhagen, Denmark.) — Acta zool. (Stockh.) 61(1): 39–49. The rete tibiotarsale is the main arterio-venous heat exchange system in the hind limb of the Mallard. A large arterial shunt and a venous shunt allow the blood to by-pass the rete. These shunt vessels must be able to constrict so as to direct the blood to the rete when heat conservation is needed. Using ordinary histological methods and the technique of Falck and Hillarp for demonstration of biogenic monoamines, it was shown that the arterial shunt is more muscular and receives a more dense adrenergic innervation than adjacent segments of the same vessel. Perfusion with noradrenaline before fixation revealed that the arterial shunt was able to reduce its lumen to near closure. No structure, in the ordinary sense of a sphincter, was found in the shunt vein but adrenergic nerves were scattered throughout the tunica media at the base of venous valves, suggesting that a sphincteric action at these sites is possible.     

Atrial septostomy benefits severe pulmonary hypertension patients by increase of left ventricular preload reserve

At present, it is unknown why patients suffering from severe pulmonary hypertension (PH) benefit from atrial septostomy (AS). Suggested mechanisms include enhanced filling of the left ventricle, reduction of right ventricular preload, increased oxygen availability in the peripheral tissue, or a combination. A multiscale computational model of the cardiovascular system was used to assess the effects of AS in PH. Our model simulates beat-to-beat dynamics of the four cardiac chambers with valves and the systemic and pulmonary circulations, including an atrial septal defect (ASD). Oxygen saturation was computed for each model compartment. The acute effect of AS on systemic flow and oxygen delivery in PH was assessed by a series of simulations with combinations of different ASD diameters, pulmonary flows, and degrees of PH. In addition, blood pressures at rest and during exercise were compared between circulations with PH before and after AS. If PH did not result in a right atrial pressure exceeding the left one, AS caused a left-to-right shunt flow that resulted in decreased oxygenation and a further increase of right ventricular pump load. Only in the case of severe PH a right-to-left shunt flow occurred during exercise, which improved left ventricular preload reserve and maintained blood pressure but did not improve oxygenation. AS only improves symptoms of right heart failure in patients with severe PH if net right-to-left shunt flow occurs during exercise. This flow enhances left ventricular filling, allows blood pressure maintenance, but does not increase oxygen availability in the peripheral tissue.     

Study of dynamic microcirculatory problems in ‘blackfoot disease’ -emphasizing its differences from arteriosclerosis

The cutaneous microcirculation can be divided into thermoregulatory shunt vessels and nutritive skin capillaries. Flux in nonnutritional shunt vessels dominates the signal recorded by the laser Doppler flowmeter. Computerized videophotometric capillaroscopy is a sensitive method for assessing cutaneous nutritive microcirculation. Using patients with blackfoot disease and arteriosclerosis as disease models, we evaluated the sensitivity and clinical usefulness of these relatively new techniques for peripheral vascular disorders. In blackfoot disease, blood flux measured by the laser Doppler flowmeter in the affected toe was lower than that in the nonaffected toe. In symptom-free fingers, blood flow was not significantly different between blackfoot disease and arteriosclerosis. However, blood flow in both diseases was lower than that of the control group. Patients who had the same status of thermoregulatory flow and eyeground arteriosclerotic classification underwent a 1-min arterial occlusion of the digits. The postocclusive reactive hyperemia response (PRH) of nailfold capillary loops was evaluated. All parameters for PRH for the cutaneous nutrient microcirculation including resting capillary blood cell velocity (rCBV), peak capillary blood cell velocity (pCBV) and time to pCBV were more significantly disturbed in the blackfoot disease group than in the arteriosclerotic group. On the basis of the results of this study, dynamic capillaroscopy provides a new approach for the early detection of circulatory disturbances resulting from different mechanisms.     

Oxygen diffusion from capillary layers with concurrent flow

Oxygen transport from capillary layers with concurrent flow is considered for symmetric and asymmetric distributions of oxygen concentration between the layers. The analysis is based on the solution previously obtained by the author [1]. Solutions for the symmetric case are shown to be very close to the corresponding solutions of the Krogh cylinder model. Asymmetry in oxygen distribution is introduced systematically by considering different velocities of blood in the alternate capillary layers, different inlet capillary oxygen tensions, and different capillary hematocrits. It is shown that increase of the degree of asymmetry leads to diminution of the mean oxygen tension.     

Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries

Dhadwal, Amit, Barry Wiggs, Claire M. Doerschuk, and RogerD. Kamm. Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries. J. Appl. Physiol. 83(5): 1711-1720, 1997.Atheoretical model is developed to simulate the flow of blood throughthe capillary network in a single alveolar septum. The objective is tostudy the influence of random variability in capillary dimension andcompliance on flow patterns and pressures within the network. Thecapillary bed is represented as an interconnected rectangular grid ofcapillary segments and junctions; blood flow is produced by applying apressure gradient across the network. Preferred flow channels are shownto be a natural consequence of random anatomic variability, the effectof which is accentuated at low transcapillary pressures. Thedistribution of pressure drops across single capillary segments widenswith increasing network variability and decreasing capillary transmuralpressure. Blockage of one capillary segment causes the pressure dropacross that segment to increase by 60%, but the increase falls to<10% at a distance of three segments. The factors that causenonuniform capillary blood flow through the capillary network arediscussed.

     

Progression of Diabetic Capillary Occlusion: A Model

An explanatory computational model is developed of the contiguous areas of retinal capillary loss which play a large role in diabetic maculapathy and diabetic retinal neovascularization. Strictly random leukocyte mediated capillary occlusion cannot explain the occurrence of large contiguous areas of retinal ischemia. Therefore occlusion of an individual capillary must increase the probability of occlusion of surrounding capillaries. A retinal perifoveal vascular sector as well as a peripheral retinal capillary network and a deleted hexagonal capillary network are modelled using Compucell3D. The perifoveal modelling produces a pattern of spreading capillary loss with associated macular edema. In the peripheral network, spreading ischemia results from the progressive loss of the ladder capillaries which connect peripheral arterioles and venules. System blood flow was elevated in the macular model before a later reduction in flow in cases with progression of capillary occlusions. Simulations differing only in initial vascular network structures but with identical dynamics for oxygen, growth factors and vascular occlusions, replicate key clinical observations of ischemia and macular edema in the posterior pole and ischemia in the retinal periphery. The simulation results also seem consistent with quantitative data on macular blood flow and qualitative data on venous oxygenation. One computational model applied to distinct capillary networks in different retinal regions yielded results comparable to clinical observations in those regions.     

Relationships between Cerebral Blood Flow Dynamics and Bioelectric Activity of the Human Brain in Experimental Acute Hypoxia

The relationships between the parameters of oxygen content in the body (hemoglobin saturation with oxygen and trancutaneous oxygen tension), central hemodynamics (cardiac output), and cerebral hemodynamics (cerebral blood flow rate) were studied during a hypoxic test (inhalation of an oxygen–nitrogen mixture containing 8% oxygen for 15 min). Special attention was paid to the relationships between the dynamics of cerebral blood flow and cerebral bioelectric activity measured by EEG parameters. It was demonstrated that the trancutaneous oxygen tension decreased to a greater extent than the hemoglobin saturation with oxygen and the cerebral blood flow increased to a greater extent than the cardiac output. The increase in cerebral blood flow and the increase in the indices and power of and EEG waves in the course of hypoxia were strongly positively correlated with each other in most subjects. However, if these parameters were considered in the series of subjects, the degree of the increase in the indices and power of and waves in different subjects was negatively correlated with the increase in the cerebral blood flow. The results are explained in terms of redistribution of blood flow in the body to provide a better oxygen supply to the brain and optimization of the ratios between the cerebral oxygen consumption and the functional load on the system of oxygen supply.