MOSAICING OF DYNAMIC MESENTERY VIDEO WITH GRADIENT BLENDING.

In biomedical imaging using video microscopy, understanding large tissue structures at cellular and finer resolution poses many image acquisition challenges including limited field-of-view and tissue dynamics during imaging. Automated mosaicing or stitching of live tissue video microscopy enables the visualization and analysis of subtle morphological structures and large scale vessel network architecture in tissues like the mesentery. But mosacing can be challenging if there are deformable, motion-blurred, textureless, feature-poor frames. Feature-based methods perform poorly in such cases for the lack of distinctive keypoints. Standard single block correlation matching strategies might not provide robust registration due to deformable content. In addition, the panorama suffers if there is motion blur present in a sequence. To handle these challenges, we propose a novel algorithm, Deformable Normalized Cross Correlation (DNCC) image matching with RANSAC to establish robust registration. Besides, to produce seamless panorama from motion-blurred frames we present gradient blending method based on image edge information. The DNCC algorithm is applied on Frog Mesentery sequences. Our result is compared with PSS/AutoStitch [1, 2] to establish the efficiency and robustness of the proposed DNCC method.

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

Endothelium-medicated control of the coronary circulation. Exercise training-induced vascular adaptations.

This review discusses the role of the endothelium in the regulation of coronary vascular function. The role of endothelium-mediated mechanisms at rest, during exercise, in exercise training-induced adaptations of coronary function and in the presence of coronary heart disease (CHD) are examined. Mechanisms of control of coronary blood flow are briefly discussed with emphasis on endothelium-mediated control of vascular resistance. The concept that the relative importance of vascular control mechanisms differs as a function of position along the coronary arterial tree is developed and discussed. Metabolic, myogenic and endothelium-mediated control systems contribute in parallel to regulating coronary blood flow. The relative importance of these mechanisms varies throughout the coronary arterial tree. Endothelium-dependent vasodilation contributes to maintenance of resting coronary blood flow but the endothelium's role in dilation of small resistance arteries, thereby increasing coronary blood flow during exercise, remains in question. In contrast, the endothelium plays an essential role in dilation of the conduit coronary arteries during exercise. Atherosclerosis and CHD convert this exercise-induced dilation to a vasoconstriction, apparently due to endothelium dysfunction. Long term increases in physical activity and exercise training alter the control of coronary blood flow. Adaptations in endothelium-mediated control play a role in these changes. However, the effects of the mode, frequency, and intensity of exercise training bouts and duration of training on adaptive changes in endothelial function have not been established. The role of the endothelium in control of the permeability characteristics of the exchange vessels in the coronary circulation is discussed. Current evidence indicates that vascular permeability is a dynamic characteristic of the vessel wall that is controlled, at least in part, by endothelium-dependent phenomena. Also, preliminary results indicate that exercise training alters microvessel permeability and the control of permeability in the coronary circulation. Further research is needed to provide clarification of the effects of exercise training on coronary endothelial control of vascular resistance and vascular permeability in atherosclerosis and CHD.

Cerebral vascular response to hypertonic fluid resuscitation in thermal injury.

The purpose of this project was to study the effects of various resuscitation fluid protocols in a systemically thermally injured rat sustaining 70% body surface area third degree burn using the pial window model in rats. The results show that there was a significant albumin leak in the cerebral vessels in both the experimental group which underwent no resuscitation fluid, as well as the experimental group that was resuscitated with Lactated Ringer's solution using the Parkland formula. When this was compared to the control group, as well as to the experimental group which received hypertonic hyperosmotic saline (HMS) boluses every hour, there was little if any leakage seen.

Morphologic analysis of the cerebral microcirculation after thermal injury and the response to fluid resuscitation.

Using the pial window model, we have previously demonstrated that there is a disruption of the blood brain barrier with distal thermal injury [1-3]. Our laboratory has shown that treatment with Lactated Ringer's Solution did not improve labeled albumin leakage. However, treatment with hypertonic hyperosmotic saline (HHS) solution post thermal injury seemed to essentially eliminate the albumin leakage in cerebral vessels. Using adult Sprague-Dawley rats and epifluorescent microscopy, the cerebral vessel size and diameter were measured, as well as the number of leukocytes rolling or adherent to the endothelium. The results show that there was significant progressive arterial dilatation over six hours in the thermally injured animals treated with HHS. There was also a significant increase in leukocyte number if the animals were thermally injured and had no resuscitation fluid or if the animals were thermally injured and underwent resuscitation fluid with Lactated Ringer's compared to either the control group or the group that was treated with HHS after thermal injury.

In vivo visualization of cerebral microcirculation in systemic thermal injury.

We present a model used to describe the effects of systemic thermal injury in cerebral permeability with the use of an open, acute pial window technique. Sprague-Dawley rats were anesthetized, and an open pial window was constructed. The area was then bathed with artificial cerebrospinal fluid with a pH adjusted to 7.4 that was heated to a constant temperature of 37 degrees C, which was allowed to circulate into a reservoir at a rate of 2 cc/min. The fluid was infused with a gas mixture of 5% carbon dioxide and 95% nitrogen. A warming blanket was placed under the animal's ventral surface, and the animal's temperature was maintained at 37 degrees C and monitored with a rectal thermal probe. Experimental animals were submerged to the xiphoid process in 100 degrees C water bath for a total of 6 seconds, which produced a 70% total body surface area third degree burn. Control animals were submerged in 37 degrees C water for 6 seconds. The animals were then injected with a constant infusion of bovine albumin coupled with fluorescein isothiocyanate. Recordings were taken every 15 minutes for 6 hours. The vascular albumin leakage was determined from the ratio of interstitium to vascular fluorescence and expressed as a percentage. The percent albumin leakage in the control group was found to be significantly different from that in the experimental group at all periods measured. The mean increase in permeability ranged from 20% at 15 minutes to 104% at 6 hours. These changes were found to be statistically significant with the use of unpaired t test at a P value of .0001. The model presented is the first to demonstrate changes in cerebral permeability after acute severe systemic thermal injury.

Physiologic regulation of capillary permeability.

The review provides an update and overview of the role of exchange microvessels in the transfer of materials between circulating blood and metabolizing tissues. Following a presentation of the basic mechanisms governing transvascular movement of water and solutes, both active and passive modulation of vascular permeability are discussed. Some of the issues under current study are highlighted and a few of the questions awaiting answers are raised.

Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments.

Whereas the glycocalyx of endothelial cells has been shown to influence solute flux from capillary microvessels, little is known about its contribution to the movement of macromolecules across the walls of other microvessels. We evaluated the hypothesis that a glycocalyx contributes resistance to protein flux measured in coronary arterioles. Apparent solute permeability (P(s)) to two proteins of different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), was determined in arterioles isolated from the hearts of 43 female Yucatan miniature swine. P(s) was assessed in arterioles with an "intact" glycocalyx under control conditions and again after suffusion with adenosine (Ado, 10(-5) M, n = 42 arterioles, N = 29 pigs). In a second set of experiments (n = 21 arterioles, N = 21 pigs) arteriolar P(s) was determined before and after perfusion with enzyme (pronase or heparinase), which was used to digest the glycocalyx. P(s) was assessed a third time on those microvessels after exposure to Ado. Consistent with the hypothesis, P(s) for PSA (P(PSA)(s)) and P(s) for alpha-lactalb (P(alpha-lactalb)(s)) increased from basal levels following enzyme treatment. Subsequent suffusion with Ado, a significant metabolite known to alter coronary vascular smooth muscle tone and permeability, resulted in a significant reduction of basal P(alpha-lactalb)(s) in both untreated and enzyme-treated arterioles. Furthermore, in untreated arterioles, P(PSA)(s) was unchanged by Ado suffusion, whereas Ado induced a pronounced reduction in P(PSA)(s) of enzyme-treated vessels. These data demonstrate that in intact coronary arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes significantly to net barrier resistance to solute flux.

Differential actions of albumin and plasma on capillary solute permeability.

We tested the hypothesis that albumin regulates the solute permeability coefficients (Ps) of individually perfused exchange microvessels of the frog mesentery by mechanisms similar to those described for hydraulic conductivity. Using unbound Evans blue dye (T-1824, mol wt 960, 1.3 nm radius) as a test solute, we demonstrated a hysteresis of PT-1824s on luminal albumin content as perfusate albumin concentration was first reduced in three steps from 1 mg/ml to zero and then returned to 1 mg/ml. PT-1824 s did not return to initial control values. The same result was found with tetramethylrhodamine isothiocyanate-alpha-lactalbumin (mol wt 14,176; 2.0 nm radius) even when the perfusate albumin concentration was increased by 10-fold to 10 mg/ml. In contrast, frog plasma at the same total protein concentration restored P alpha-lactalbumin s to values below those measured with control albumin perfusates before Ringer perfusion. Our results conform to the hypothesis that a plasma factor (possibly orosomucoid) that modifies the charge selectivity of the microvessel wall is removed from the microvessel membrane during protein-free Ringer perfusion and is not restored by reperfusion with albumin alone.

Capillary hydraulic conductivity is elevated by cGMP-dependent vasodilators.

Microvascular functions have been shown to be sensitive to agents associated with changes in cyclic nucleotide levels. The central hypothesis of the current study was that one measure of capillary exchange capacity, hydraulic conductivity (Lp), would be elevated by agents shown to elevate cellular levels of cGMP. To evaluate the hypothesis, frog mesenteric capillary Lp was measured during luminal exposure to 1) atrial natriuretic peptide (ANP), 2) the truncated atriopeptins ANP-I and ANP-III, 3) the nitrovasodilator sodium nitroprusside (SNP), 4) the cGMP phosphodiesterase inhibitor M&B 22948, and 5) methylene blue dye, both alone and in combination with ANP or SNP. ANP (100 nM) elevated Lp by 2.3 +/- 0.2-fold from control levels (n = 15); 10 nM ANP induced a 2.1 +/- 0.3-fold change (n = 8), while 10 nM ANP-III elicited a 1.7 +/- 0.4-fold change (n = 8). In contrast, Lp did not change from basal levels during 10 nM ANP-I infusion (Lp ANP-I/Lp control = 1.2 +/- 0.2; n = 14). SNP (1 microM) induced a reversible, 2.6 +/- 0.5-fold increase in Lp (n = 30) that was inhibitable by methylene blue dye (Lp SNP + MetB/Lp control = 1.1 +/- 0.1; n = 8). Methylene blue did not mask the response to 100 nM ANP (Lp ANP + MetB/Lp control = 2.1 +/- 0.5; n = 7). M&B 22948 (30 microM) increased Lp by 2.8 +/- 0.6-fold (n = 9). These data constitute strong inference that agents demonstrated to elevate cGMP also mediate an increase in capillary Lp in in situ, perfused exchange vessels.

Basal and adenosine-mediated protein flux from isolated coronary arterioles.

We have developed a new method to quantify solute flux per unit surface area and concentration gradient (JS/S delta C) from arterioles isolated from pig hearts. The apparent permeability (Ps) was assessed from measurements of JS/S delta C for two proteins, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), labeled with the fluorescent dye tetramethylrhodamine isothiocyanate at a mean hydrostatic pressure of 16 +/- 1 cmH2O. Ps for alpha-lactalb (Ps alpha-lactalb) was 16.5 +/- 4.6 x 10(-7) cm/s (mean +/- SE, N = 8 pigs), a value significantly higher than Ps for PSA (PsPSA) (7.1 +/- 1.4 x 10(-7) cm/s, N = 11 pigs, P < 0.05). Suffusion of the arterioles (44 +/- 10 microns diam; n = 48 arterioles) with 10(-5) M adenosine resulted in a 35% decrease in Ps alpha-lactalb and 29% decrease in PsPSA. Data from the present study are consistent with adenosine altering arteriole Js independently from its ability to change arteriolar caliber. One implication of these results is that changes in coronary exchange capacity reflect not only changes in flow through, but also solute permeation from, the microvasculature.

O2 modulation of single-vessel hydraulic conductance.

To investigate the response of the microvascular endothelial barrier to O2 lack, hydraulic conductivities (Lp) were compared under control and hypoxic conditions. In 29 decerebrate frogs (Rana pipiens) the mesentery was exposed and continuously superfused with room air-equilibrated frog Ringer (14 degrees C, pH 7.4). Single mesenteric microvessels (15- to 50-microm diam) were cannulated and perfused with frog Ringer containing 5-40 mg/ml bovine serum albumin and 5-10% (vol/vol) human erythrocytes. The modified Landis technique was used to measure transmural water flux per unit area (Jv/S) from marker red cell movement at pressures ranging from 15 to 45 cmH2O. In 21 vessels, a 10-min superfusion with 100% N2-equilibrated Ringer increased Lp 2.4-fold (+/- 0.72 SD) over control (P less than or equal to 0.05). In an additional six vessels, simultaneous exposure to 100% N2-equilibrated perfusate and superfusate resulted in a 10-fold (+/- 3.0) increase in Lp (P less than or equal to 0.05). In 15 of the vessels, a 10- to 75-min reexposure to control conditions decreased Lp, averaging a 77% recovery toward base line. These data support an active role for O2 in the regulation of microvascular membrane permeability, which appears to be sensitive to the severity of O2 lack. The rapid increase in capillary water conductivity following exposure to hypoxic conditions may be the initial process in the events leading to edema formation.

A direct effect of atrial peptide on arterioles of the terminal microvasculature.

Responses to atrial peptide (AP) were observed in cheek pouch arterioles of anesthetized golden hamsters (pentobarbital sodium, 70 mg/kg ip). Vessels for observation were selected on the basis of their functional status in relation to controlling the perfused capillary surface area. Local application of AP onto microvessels at a final concentration of from 7 X 10(-9) to 3 X 10(-8) M was achieved via micropipettes; responses were compared with those produced by application of vehicle only. Individual vessels were observed during control conditions and during vasoconstriction of the microvascular bed induced via either equilibration of the superfusate with 10% oxygen or addition to the superfusate of 5 X 10(-7) M norepinephrine (NE). Compared with responses induced by the vehicle in each condition, AP produced a statistically significant dilation during control conditions, and during raised PO2, but no statistically significant effect during NE exposure. Mean diameter changes were from a base line of 27.8 +/- 4.7 to 29.7 +/- 4.5 microns with vehicle and 33.2 +/- 4.4 microns with AP in controls; from 22.7 +/- 4.6 to 25.8 +/- 4.5 microns with vehicle and 30.1 +/- 4.7 microns with AP during raised PO2; and from 25.2 +/- 4.7 to 29.8 +/- 5.4 microns with vehicle and 33.5 +/- 4.9 microns with AP during NE exposure. The data show that arterioles of the terminal microvasculature can respond selectively to AP.

Differential sensitivity of exchange vessel hydraulic conductivity to atrial natriuretic peptide.

Acute plasma volume reduction by atrial natriuretic peptide (ANP) may be mediated, at least in part, by increased exchange vessel water conductivity (Lp). The present study tests the hypothesis that physiological levels of ANP acutely and reversibly elevate single capillary Lp. Paired, in situ measurements of Lp were obtained using the modified Landis technique in individually perfused mesenteric capillaries of the frog, Rana pipiens. Control Lp measurements ranged from 0.1 to 40 x 10(-7) cm.s-1.cmH2O-1 (with a median value of 2.5 x 10(-7] in 81 microvessels perfused with frog Ringer solution containing dialyzed bovine serum albumin. Vessels were recannulated and perfused with one or more concentrations of human ANP (hANP) spanning the physiopathological range: 0.01, 0.1, 1, 10, and 100 nM. When possible, a final recannulation and Lp determination was performed in the absence of hANP. A median 2.2-fold rapid Lp increase was observed compared with control at each peptide concentration. Lp changes persisted for the duration of exposure to hANP, returning to control levels on withdrawal of the peptide. True and venular capillaries exhibited similar Lp responses: median 2.2- and 2.4-fold elevations, respectively. Sixty percent of true and venular capillaries exhibited twofold or greater hANP-induced increases in Lp, whereas only 25% of arteriolar capillaries exhibited such a response. Thus a differential sensitivity to the peptide exists across the exchange vessel network. The observations of this study demonstrate that physiological levels of ANP are capable of modulating exchange vessel Lp, one means by which the peptide may acutely alter plasma volume.(ABSTRACT TRUNCATED AT 250 WORDS)

ANP increases capillary permeability to protein independent of perfusate protein composition.

In further studying hormonal regulation of microvascular exchange, we tested two hypotheses: 1) atrial natriuretic peptide (ANP) would increase capillary permeability to protein and 2) the actions of ANP on capillary permeability would be independent of the perfusate protein composition. These hypotheses were tested by assessing in situ capillary transport coefficients: hydraulic conductivity (Lp), apparent and diffusive permeabilities (Ps and Psd, respectively) to the protein alpha-lactalbumine, and convective coupling [Lp(1-sigma)] in mesenteric capillaries in cerebrally pithed frogs (Rana pipiens). The transport coefficients were determined in the absence and presence of frog ANP (fANP) during perfusion with either frog plasma or bovine serum albumin (BSA). Control Lp was 1.8-fold greater in vessels perfused with BSA compared with plasma. In the presence of 10 or 100 nM fANP, Lp was increased by approximately 3.5-fold in plasma-perfused vessels and approximately 2-fold in BSA-perfused vessels from their respective controls. Control Ps was 4.2-fold higher in vessels perfused with BSA compared with plasma. Despite the differences in control permeability, the increase in Ps by fANP was of similar magnitude (2- to 3-fold) for both protein perfusates. Analysis of the pressure-dependent alpha-lactalbumin flux suggested that the increase in capillary permeability induced by fANP is consistent with fANP increasing permeability without altering the selectivity of the capillary barrier.

Effect of superfusate albumin on single capillary hydraulic conductivity.

It has been proposed that albumin interacts with the endothelial cell surface to form part of the molecular filter at the capillary wall. Previous investigations of the "protein effect" have been limited to sites accessible to albumin from the capillary lumen. We tested for a specific interaction of albumin with the ablumenal surface of the capillary wall. The hydraulic conductivity (Lp) of single capillaries in frog mesentery, perfused initially with albumin-free Ringer, was reduced when albumin (concentration greater than 1 mg/ml) was added to the superfusate (mean fractional reduction 0.56 +/- 0.05 SE, n = 15). A similar reduction was measured when the mesothelial barrier to water and solute flows between the superfusate and the albuminal surface of the capillary was destroyed. When the albumin was extensively dialyzed against Ringer to remove small vasoactive molecules, no change in the fractional reduction of Lp was observed. Lp was reduced to a minimum value in any capillary when the albumin concentration on both sides of the capillary wall was greater than 1 mg/ml. Our data conform to the hypothesis that albumin modifies structures throughout the capillary wall to maintain normal permeability. We predict that extravascular albumin reduces the ability of a Ringer perfusate to increase permeability in many isolated perfused organs.

Capillary permeability: atrial peptide action is independent of "protein effect".

Perfusion of exchange microvessels with the vasoactive hormone, atrial natriuretic peptide (AP), acutely and reversibly elevates hydraulic conductivity (Lp) by mechanisms that are, as yet, unknown. This, the first of two studies to characterize AP responses when perfusate composition was altered, specifically focuses on the action of AP when perfusate albumin was lowered to change the transcapillary barrier properties for water by passive mechanisms (protein effect). Perfusion of frog (Rana pipiens) mesenteric microvessels with 1 nM AP in 10 mg/ml bovine serum albumin (BSA) elevated Lp by a median 2.1-fold (range 1.2-2.7, n = 13) from control levels (10 mg/ml BSA). Reduction of perfusate albumin from 10 to 1 mg/ml elicited a small rise in Lp (1.8-fold, n = 10); Lp rose a further 2.1-fold (n = 6) when 1 nM AP was added to 1 mg/ml BSA. Likewise, protein-free perfusion elevated Lp from a median 2.2 to 5.1 X 10(-7) cm.s-1.cmH2O-1 (n = 11); 1 nM AP in protein-free perfusate elevated Lp a further 2.1-fold (n = 8). Thus, regardless of protein content, the response to the peptide was a consistent, twofold increase in exchange vessel Lp (n = 27). These data are consistent with the suggestion that the AP-activated rise in Lp (twofold) occurs via an increase in the effective area of the transcapillary pathway for water without influencing the selectivity properties of the paracellular, albumin-sensitive portion of the barrier.

Capillary permeability: an albumin component attenuates active changes in Lp.

The mechanisms whereby atrial natriuretic peptide (AP) acutely and reversibly elevates hydraulic conductivity (Lp) are not known. This is the second of two studies of the influence of perfusate albumin composition on AP alterations in capillary Lp. In this study, we investigated the effect of dialysis of the perfusate albumin. A 2.2-fold increase in frog (Rana pipiens) mesenteric microvessel Lp occurred when 100 nM AP was added to the control perfusate containing dialyzed, crystallized bovine serum albumin (DXL-BSA 10 mg/ml; n = 20). By contrast, Lp was unchanged by 100 nM AP in 10 mg/ml untreated, crystallized BSA (XL-BSA; n = 8). The response to AP was unaltered at DXL-BSA contents of 10, 20, or 30 mg/ml (2.4-, 2.2-, and 3.1-fold, respectively; n = 8). Dialysis of the albumin, per se, did not influence control Lp (LpXL-BSA/LpDXL-BSA = 1.0; n = 5). The receptor-independent nitrovasodilator, sodium nitroprusside (SNP; 1 microM) elevated Lp by 1.7-fold in DXL-BSA (n = 30). The response was abolished in XL-BSA (n = 8). We conclude that small hydrophilic albumin-associated substances antagonize AP- and SNP-induced elevations of exchange microvessel hydraulic conductance without interfering with albumin's role in the maintenance of normal exchange vessel permeability.

Evidence for cholinergic regulation of microvessel hydraulic conductance during tissue hypoxia.

Cholinergic regulation of single-vessel hydraulic conductivity (Lp) during normoxia and hypoxia was tested in single mesenteric vessels of pithed frogs (Rana pipiens). Capillaries were cannulated in situ and perfused with frog Ringer's solution containing 10 mg/ml albumin and human erythrocytes while the mesentery was continuously superfused with frog Ringer's solution (15 degrees C). Lp was first measured under normoxic (room air equilibrated) conditions by the modified Landis microocclusion method. Repeated measurements of filtration coefficient under control conditions, for periods up to 80 minutes, demonstrated that Lp did not change with time in normoxic vessels (n = 18). After initial control measurement (Lpo), perfusion with 1 microM acetylcholine increased Lp by 4.6 +/- 1.0-fold (mean +/- SEM, n = 6). The response to acetylcholine was antagonized by the addition of 10 microns atropine to the perfusate (Lp/Lpo = 1.8 +/- 0.4). Perfusion with atropine alone reduced Lp in three of six capillaries Lp/Lpo = 0.56 +/- 0.04); Lp in the remaining three vessels was unaffected. Tissue hypoxia was simulated by exposing the mesentery to deoxygenated superfusate Po2 less than or equal to 10 mm Hg) for 10-15 minutes. Tissue hypoxia had no effect on Lp in atropine-treated vessels (n = 8). Without atropine, tissue hypoxia increased Lp by 2.3 +/- 0.7-fold, whereas the addition of atropine completely antagonized this response (n = 5). In contrast to the inhibitory action of atropine during tissue hypoxia, Lp rose 5.2 +/- 1.6-fold (n = 4) in vessels simultaneously exposed to deoxygenated perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)