High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

Study of blood flow in microvessels using biospeckle dynamics

The hydrodynamic properties of the blood plasma flow in smallest microvessels have been investigated. It has been shown that velocity distribution in such flows essentially differs from the Poseuille flow. The interrelations between the optical parameters and hydrodynamic characteristics of blood microflow have been analyzed. A new method for in vivo measurement of blood plasma rate in small microvessels is proposed, which uses in vivo microscopy in combination with speckle microscopy.     

Two-photon imaging of calcium in virally transfected striate cortical neurons of behaving monkey

Two-photon scanning microscopy has advanced our understanding of neural signaling in non-mammalian species and mammals. Various developments are needed to perform two-photon scanning microscopy over prolonged periods in non-human primates performing a behavioral task. In striate cortex in two macaque monkeys, cortical neurons were transfected with a genetically encoded fluorescent calcium sensor, memTNXL, using AAV1 as a viral vector. By constructing an extremely rigid and stable apparatus holding both the two-photon scanning microscope and the monkey's head, single neurons were imaged at high magnification for prolonged periods with minimal motion artifacts for up to ten months. Structural images of single neurons were obtained at high magnification. Changes in calcium during visual stimulation were measured as the monkeys performed a fixation task. Overall, functional responses and orientation tuning curves were obtained in 18.8% of the 234 labeled and imaged neurons. This demonstrated that the two-photon scanning microscopy can be successfully obtained in behaving primates.     

Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain

An understanding of the logic of odor perception requires a functional analysis of odor-evoked patterns of activity in neural assemblies in the brain. We have developed a sensitive imaging system in the Drosophila brain that couples two-photon microscopy with the specific expression of the calcium-sensitive fluorescent protein, G-CaMP. At natural odor concentration, each odor elicits a distinct and sparse spatial pattern of activity in the antennal lobe that is conserved in different flies. Patterns of glomerular activity are similar upon imaging of sensory and projection neurons, suggesting the faithful transmission of sensory input to higher brain centers. Finally, we demonstrate that the response pattern of a given glomerulus is a function of the specificity of a single odorant receptor. The development of this imaging system affords an opportunity to monitor activity in defined neurons throughout the fly brain with high sensitivity and excellent spatial resolution.     

A study of blood flow in microvessels using biospeckle dynamics

Hydrodynamic properties of blood flows in small microvessels and the patterns of scattering of focused laser beams in such flows were studied. The processes of formation of dynamic biospeckles are considered. The relationship between the optical parameters and hydrodynamic characteristics of blood microflow are analyzed. A new method is proposed for measureming the plasma rate in small microvessels with the use of in vivo microscopy in combination with speckle microscopy.     

Evaluation of laser-Doppler perfusion imaging for measurement of blood flow in cortical bone.

Most techniques currently available to measure blood flow in bone are time consuming and require destruction of the tissue, but laser-Doppler technology offers a less invasive method. This study assessed the utility of laser-Doppler perfusion imaging (LDI) to measure perfusion in cortical bone. Twelve mature New Zealand White rabbits were assigned to one of three groups: normal control, constriction (norepinephrine), or dilatation (nitroprusside). The left and right medial tibiae were consecutively scanned at red (634-nm) and near-infrared (810-nm) wavelengths to examine the repeatability of LDI output. The pharmacological intervention groups were injected with the respective drug, and LDI measurements at 810 nm were obtained concurrently with colored microsphere-determined flow in all of the groups. LDI effectively quantified blood flow in cortical bone and detected physiologically induced changes in perfusion. A significant positive correlation was found between microsphere-determined flow and LDI output (r = 0.6, P < 0.05). Repeatability of consecutive LDI measurements was within 5%. The effectiveness of LDI to measure perfusion in bone suggests this method has potential for investigating the role of blood flow in bone metabolism and remodeling.     

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.     

An impulse-digital method of measuring blood flow velocity in microvessels]

The photometric trigger method described was used for determining the velocity of erythrocytes. It allows to take continuous erythrocyte velocity and cell flux measurements in the microvessels. Application of the impulse-digital chronometry of the intervals of the erythrocyte transit time ensures direct transformation of the measuring trigger signals into a digital code. The results of testing the device on the microvessels of frog mesentery are given.     

Analysis of pulmonary arterial pressure profile after occlusion of pulsatile blood flow

In isolated canine lung lobes perfused with a pulsatile pump, arterial occlusions were performed and the postocclusion arterial pressure profiles were analyzed to estimate the pulmonary capillary pressure. A solenoid valve interposed between the pump and the lobar artery was used to perform arterial occlusions at several instants equally distributed within a pressure cycle. Double occlusions were also accomplished by simultaneously activating the solenoid valve and clamping the venous outflow of the lung lobe. To analyze an arterial occlusion pressure profile, we computed the best monoexponential fit of the pressure decay over a short period of time after the occlusion maneuvers. Two estimates of the capillary pressure were derived from this analysis: 1) the extrapolation of the exponential fit to the instant of occlusion, and 2) the point at which the recorded pressure decay curve merges with the exponential fit. The pressures thus determined were compared with the double occlusion pressure that provided an independent estimate of the pulmonary capillary pressure. Our results show that, under a wide range of conditions, the estimates of the capillary pressure obtained from the arterial occlusion data are nearly equal to the double occlusion pressures. Additionally, we estimated the capillary pressure variations within a pressure cycle by examining the occlusion pressures sampled at different instants of the cycle. The pulsatility of the pulmonary microvascular pressure varied with the pump frequency as well as the state of arterial and venous vasoaction. These variations are consistent with the representation of the lung vasculature as a low-pass filter.     

Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.     

Systemic blood flow to the lung after bronchial artery occlusion in anesthetized sheep.

To compare the effectiveness of different embolizing agents in reducing or redistributing bronchial arterial blood flow, we measured systemic blood flow to the right lung and trachea in anesthetized sheep by use of the radioactive microsphere method before and 1 h after occlusion of the bronchoesophageal artery (BEA) as follows: injection of 4 ml ethanol (ETOH) into BEA (group 1, n = 5), injection of approximately 0.5 g polyvinyl alcohol particles (PVA) into BEA (group 2, n = 5), or ligation of BEA (group 3, n = 5). After occlusion, angiography showed complete obstruction of the bronchial vessels. There were no changes in tracheal blood flow in any of the groups. Injection of ETOH produced a 75 +/- 14% (SD) reduction in flow to the middle lobe (P less than 0.02) and a 75 +/- 13% reduction to the caudal lobe (P less than 0.01), whereas injection of PVA produced a smaller reduction in flow to these two lobes (41 +/- 66 and 51 +/- 54%, respectively). After BEA ligation there was a 52 +/- 29% reduction in flow to the middle lobe and a 53 +/- 38% reduction to the caudal lobe (P less than 0.05). This study has significant implications both clinically and experimentally; it illustrates the importance of airway collateral circulation, in that apparently complete radiological obstruction of the BEA does not necessarily mean complete obstruction of systemic blood flow. We also conclude that, in experimental studies in which the role of the bronchial circulation in airway pathophysiology is examined, ETOH is the agent of choice.     

Prior exercise training increases collateral-dependent blood flow in rats after acute femoral artery occlusion

We evaluated whether prior training would improve collateral blood flow (BF) to the calf muscles after acute-onset occlusion of the femoral artery. Exercise training was performed in the absence of any vascular occlusion. Adult male Sprague-Dawley rats ( approximately 325 g) were kept sedentary (n = 14), limited to cage activity, or exercise trained (n = 14) for 6 wk by treadmill running. Early in the day of measurement, animals were surgically prepared for BF determination, and the femoral arteries were occluded bilaterally. Four to five hours later, collateral BF was determined twice during treadmill running with the use of (141)Ce and (85)Sr microspheres: first, at a demanding speed and, second, after a brief rest and at a higher speed. The absence of any further increase in BF at the higher speed indicated that maximal collateral BF was measured. Prior training increased calf muscle BF by approximately 70% compared with sedentary animals; however, absolute BF remained below values previously observed in animals with a well-developed collateral vascular tree. Thus prior training appeared to optimize the use of the existing collateral circuit. This implies that altered vasoresponsiveness induced in normal nonoccluded vessels with exercise training serves to improve collateral BF to the periphery.     

Two-photon live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche

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Shear stress and intravascular pressure effects on vascular dynamics: two-phase blood flow in elastic microvessels accounting for the passive stresses

Biomechanics and Modeling in Mechanobiology - We study the steady hemodynamics in physiological elastic microvessels proposing an advanced fluid–structure interaction model. The arteriolar...     

Velocity profiles of blood flow in microvessels measured by ten channels' dual-sensor method

Ten channels' dual-sensor method newly developed was applied to the measurement of velocity profiles in arterioles and venules in the rat mesentery. In some experiments, red blood cells (RBC), labelled in vitro with a fluorescein isothiocyanate (FITC), were injected to compare the velocity profiles obtained by the RBC visualization technique with those measured by the dual-sensor method. It was found that the velocity profile of the FITC labelled RBC in straight microvessels was blunt as compared to a parabola. The centerline velocity measured by the dual-sensor method was smaller than that of the FITC labelled RBC by about 20%. The velocity profiles were also measured at the curved arterioles and venules as well as at the bifurcation and the confluence. It was found that the velocities were higher along the inner wall at the curved portion and along the outer wall at the bifurcation of arterioles.