Contrast-enhanced ultrasound measurement of microvascular perfusion relevant to nutrient and hormone delivery in skeletal muscle: a model study in vitro

Contrast-enhanced ultrasound (CEU) has been used to measure muscle microvascular perfusion in vivo in response to exercise and insulin. In the present study we address whether CEU measurement of capillary volume is influenced by bulk flow and if measured capillary filling rate allows discrimination of different flow pattern changes within muscle. Three in vitro models were used: (i) bulk flow rate was varied within a single length of capillary tubing; (ii) at constant bulk flow, capillary volume was increased 3-fold by joining lengths of capillary in series, and compared to a single length; and (iii) at constant bulk flow, capillary volume was increased by sharing flow between a number of lengths of identical capillaries in parallel. The contrast medium for CEU was gas-filled albumin microbubbles. Pulsing interval (time) versus acoustic-intensity curves were constructed and from these, capillary volume and capillary filling rate were calculated. CEU estimates of capillary volume were not affected by changes in bulk flow. Furthermore, as CEU estimates of capillary volume increased, measures of capillary filling rate decreased, regardless of whether capillaries were connected in series or parallel. Therefore, CEU can detect a change in filling rate of the microvascular volume under measurement, but it can not be used to discriminate between different flow patterns within muscle that might account for capillary recruitment in vivo.     

Protamine inhibits capillary formation in growing rat hearts

Various indices of capillary supply to the rat heart were studied in neonatal rats injected for 2 or 4 weeks with protamine sulfate in saline (subcutaneously, 60 mg/kg body weight, 2 times/day). Cardiac capillarization was evaluated not only by traditional indices for the capillary supply, such as mean capillary density and myocyte-to-capillary ratio, but also by a more advanced morphometric method of capillary domains. This method allows the estimation of both the average radius of the Krogh tissue cylinder and its variability, which reflects the heterogeneity of capillary spacing found to be an independent morphological determinant of oxygen diffusion in the tissue. The results were evaluated with respect to regional differences (subendocardial vs. middle section), age differences, and the effect of protamine. No regional differences in capillary supply were found in this experimental situation. Hearts from older rats had significantly decreased capillary supply, expressed as lower capillary density, larger capillary domains, and greater radius of the tissue cylinder. On the other hand, the heterogeneity of capillary spacing decreased significantly with age. Protamine-injected animals, when compared to their control littermates, had a significantly higher cell-to-capillary ratio, lower capillary density, larger capillary domains, greater radius of the tissue cylinder, and larger variability in capillary spacing. Thus, protamine was effective in impeding rapid capillary growth in the hearts from rats in the early postnatal period. Close to half of all the existing capillaries in the adult rat hearts are formed during the first 3-4 postnatal weeks.     

Sequential nailfold capillary microscopy in scleroderma and related disorders.

Sequential nailfold capillary microscopy was carried out monthly for seven months in seven patients (four with scleroderma, one with dermatomyositis, one with mixed connective tissue disease, and one with limited connective tissue disease). Progressive enlargement of some capillary loops was observed, with a number becoming obliterated, leaving avascular areas. Extravasation from capillaries usually preceded capillary loss. These observations have shown the progressive nature of the nailfold capillary abnormalities associated with these disorders and suggest that capillary enlargement is the result of injury, rather than compensation for capillary loss.     

The microvasculature in skeletal muscle. IV. A model of the capillary network

A reconstruction of the capillary network in the rat spinotrapezius muscle was carried out using carbon-filled specimens. A new branching schema for the capillaries, consisting of individual capillary bundles, is proposed. Each bundle is provided with blood through transverse arterioles and drained by collecting venules. A capillary bundle may be divided into capillary bundle elements consisting of a transverse arteriole, a collecting venule, and the intermediate capillary network. A set of seven independent network parameters are proposed and measurements are provided for two rat strains: Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). A comparison of network parameters indicates a multifaceted difference between the strains. In SHR the number of capillary cross-connections is reduced, individual capillary lengths are greater, and vessel diameters are slightly larger.     

Stereological investigation in the cerebral cortex of aging subjects (author's transl)]

Involving cortical regions, capillaries of the human cerebrum of two 19 and 27 years old men, a 69 years old woman and a 72 years old man were stereologically investigated by optical-electronic image-analysis. The cortical capillary net work was demonstrated by the alkaline phosphatase activity. Each cortex region comprised a determination of the stereological parameters diameter, projected area, specific surface area, capillary distances in linear direction of TV-lines and total length per unit cortex volume. A comparison between different cortex regions revealed a good correlation between increased values of the diameter and the projected area, a decreased specific surface area and diminished capillary distances, which entail a shortened distance of oxygen diffusion through the cortical tissue. During aging a diminished capillary surface area, which results from increased values of diameter and projected area is compensated by shortened capillary distances. Presumably an augmented capillary length is due to a condensation of the capillary net per unit cortex tissue. The behaviour of the registered stereological parameters seems to be an accommodation of the capillary net in the human cerebrum to metabolic and circulatory changes during aging.     

Effect of endothelin and endothelin receptor blockade on capillary permeability in experimental pancreatitis

BACKGROUND: Capillary leakage with fluid loss into the third space contributes to many of the early systemic complications in severe acute pancreatitis. There has been increasing interest in endothelin as one of the factors affecting capillary permeability. AIM: To elucidate further the role of endothelin in the development of capillary leakage in acute pancreatitis by investigating the effect of exogenous endothelin administration and endothelin receptor blockade in sham operated animals and two models of acute pancreatitis. METHODS: Determination of capillary permeability in the pancreas and colonic mucosa by quantifying extravasation of fluorescein labelled dextran using a novel computer assisted video image analysis system. RESULTS: Pancreatic and colonic capillary permeability increased stepwise from mild to severe acute pancreatitis. Endothelin increased pancreatic and colonic capillary permeability in healthy animals and animals with mild acute pancreatitis but had no additional adverse effect in severe acute pancreatitis. Endothelin receptor blockade decreased pancreatic capillary permeability in sham operated rats but had no effect on the colon. In mild and severe acute pancreatitis, endothelin receptor blockade stabilised increased capillary permeability in both the pancreas and colon. CONCLUSIONS: Endothelin plays an important role in mediating capillary permeability in the pancreas. In severe pancreatitis, it increases capillary permeability even outside the pancreas, thereby contributing to capillary leakage. Endothelin receptor blockade significantly reduces capillary permeability in acute pancreatitis both in and outside the pancreas, suggesting a therapeutic approach to counteract capillary leakage in severe acute pancreatitis.     

The Design of Cement Mortar with Low Capillary Suction: Understanding the Effect of Fine Aggregate and Sodium Silicate

The article presents the results of research that was carried out in order to analyze the capillary suction of cement mortar. Capillary suction is a common process that occurs when porous material is in free contact with moisture. The result of high capillary suction may be excessive moisture in buildings, and it is therefore important to limit the causes of such moisture. The main aim of the presented research is to show the influence of sodium silicate (in various proportions), as well as the quantity of aggregate, on capillary suction. Three different types of cement mortar and one type of fine aggregates were analyzed in the research. At the beginning, the capillary suction of the aggregates was analyzed. Afterwards, nine cement mortar bars were made, which were then used to examine the capillary suction. As a result of this study, it was proved that M15 cement mortar with basalt fine aggregate and a higher proportion of sodium silicate was the mortar with the lowest capillary suction. It was found that M15 cement mortar with basalt fine aggregate and a higher proportion of sodium silicate had 39 mm of capillary suction after 120 h of being immersed in water. M5 cement mortar without sodium silicate had the highest index of capillary suction, which shows that adding sodium silicate to cement mortar can significantly reduce capillary suction.     

Requirement of arteriovenular pairing for increased capillary filtration during acute inflammation

OBJECTIVE: The purpose of this study was to determine whether the leukocyte chemoattractant N-formyl methionyl-leucyl-phenylalanine (FMLP) increases capillary fluid filtration rate (Jv/S), and if so, to determine whether the mechanism involves arteriovenular communication. METHODS: Intravital microscopy was used in the rat mesentery to measure capillary Jv/S by a micro-occlusion technique. FMLP was locally superfused through a micropipette near vessels in three different arrangements: 1) near a venule in close pairing with the arteriole feeding the monitored capillary, 2) near an unpaired arteriole feeding the capillary, or 3) near an unpaired venule draining the capillary. In separate experiments, arteriolar hydrostatic pressure (downstream of arteriovenular pairing sites exposed to FMLP) was measured with a micropressure system. RESULTS: FMLP increased capillary Jv/S when applied to arteriovenular pairing sites, but not when applied to unpaired arterioles or venules. Micropressure measurements in paired arterioles remained essentially constant during FMLP exposure, suggesting that the increase in Jv/S is primarily due to increased capillary permeability. CONCLUSIONS: With FMLP applied locally to stimulate leukocytes, arteriovenular pairing is required to observe an increase in capillary filtration rate.     

Capillary pressure gradients in cremaster muscle of normotensive and spontaneously hypertensive rats

The purpose of this investigation is to test the hypothesis that capillary pressure gradients are elevated in spontaneously hypertensive rats (SHR) and to determine the mechanism for the elevation. The cremaster muscle was prepared for microscopic examination under chloralose-urethane anesthesia in seven SHR and eight Wistar-Kyoto (WKY) rats 4-6 weeks of age. Capillary hematocrit, diameter, and red cell velocity were measured. Capillary flow induced by a time-varying pressure gradient was treated mathematically. A finite Hankel transformation was applied to the Navier-Stokes equation for capillary vessels. The solution was expressed as a Fourier-Bessel series, and the fluctuation of capillary flow induced by a time-varying pressure gradient was studied. It was shown that if the velocity fluctuation depended only on the pressure gradient, then the velocity fluctuation would be diminished almost instantly after the capillary started to flow. Capillary pressure gradient and shear stress were evaluated according to two different flow models, Newtonian and Casson. The capillary viscosity was obtained from the capillary hematocrit based on the empirical correlation of viscosity vs hematocrit. Calculations based on both flow models indicate that the capillary pressure gradient and shear stress of SHR is higher than in WKY, especially in vessels near 6 microns in diameter. The elevated pressure gradient is due to a combination of reduced capillary density, causing a higher red cell velocity and a tendency toward smaller capillary diameters in the SHR. Capillary hematocrit and viscosity were not elevated in the SHR.     

Familial pulmonary capillary hemangiomatosis resulting in primary pulmonary hypertension

We describe the first cases of familial pulmonary capillary hemangiomatosis, a disorder in which capillaries in the lungs proliferate. Three siblings died from primary pulmonary hypertension. One developed pulmonary congestion preterminally after vasodilator treatment. The inheritance pattern seems autosomal recessive. Lung specimens obtained in two siblings showed extensive pulmonary capillary hemangiomatosis, with normal capillaries proliferating into veins and alveoli. Including our patients, four of the nine patients with pulmonary capillary hemangiomatosis have presented with the clinical picture of primary pulmonary hypertension. Thus, pulmonary capillary hemangiomatosis should be considered as a histologic pattern of primary pulmonary hypertension. Most other cases of pulmonary capillary hemangiomatosis have been similar to pulmonary veno-occlusive disease. Recently, disorders involving the proliferation of cytologically normal capillaries have been termed angiogenic diseases. Pulmonary capillary hemangiomatosis may be an angiogenic disease.     

Nailfold capillary density as a possible indicator of pulmonary capillary loss in systemic lupus erythematosus but not in mixed connective tissue disease.

Nailfold capillary density was measured in 24 patients with systemic lupus erythematosus (SLE), 14 with mixed connective tissue disease (MCTD) and 21 healthy subjects. Pulmonary function tests were performed on all subjects and needle muscle biopsies on 12 patients with SLE and 9 with MCTD. A significant correlation was documented between nailfold capillary density and pulmonary gas transfer (KCO) in patients with SLE (p less than 0.001) but not in patients with MCTD. This suggests that in SLE poor gas transfer may be dependent on alveolar capillary loss and that nailfold capillary density may be a good indicator of alveolar capillary density. There was no significant correlation between skeletal muscle fiber atrophy and nailfold capillary density in SLE or MCTD. Additional studies to optimize the nailfold capillary counting method are described.     

Obesity, insulin resistance, and capillary recruitment

OBJECTIVE: Insulin has vascular actions within the skeletal muscle microcirculation (capillary recruitment) that enhance its own access and that of glucose to the muscle cells. Obesity and insulin resistance are associated with dysregulated vascular function within muscle and a loss of insulin-mediated capillary recruitment. Furthermore, agents that impair insulin's vascular actions to recruit capillaries lead to acute insulin resistance in terms of muscle glucose uptake. Together these data suggest a strong connection between the loss of insulin-mediated capillary recruitment and the development of insulin resistance. This review examines the mechanisms involved in insulin-mediated capillary recruitment and the vascular defects associated with obesity and insulin resistance that may impair the capillary recruiting process. Understanding the mechanisms of insulin-mediated capillary recruitment and its impairment may lead to new treatment avenues to prevent the progression of obesity to diabetes.     

Maintenance of heterogeneity of capillary spacing is essential for adequate oxygenation in the soleus muscle of the growing rat

OBJECTIVES: Normal muscle growth is accompanied by capillary proliferation, which usually lags behind the increase in muscle size, causing a decline in mean capillary density (CD). It is not known, however, how the capillary distribution is affected and what impact it has on the oxygenation of the muscle. METHODS: The capillarization of soleus muscles of rats (64-425 g) was determined with the method of capillary domains. As well as quantifying CD, capillary to fiber ratio (C:F), and fiber size, this method provides a measure of the heterogeneity of capillary spacing. Capillary locations were used to mathematically model oxygenation levels within the muscle. RESULTS: The increase in muscle mass was largely attributable to 5-fold increase in fiber size, accompanied by a more than 3-fold rise in C:F. The mismatch between rates of angiogenesis and muscle growth resulted in a decrease in CD. However, the heterogeneity of capillary spacing was unaffected (heterogeneity index logRSD: 0.091 +/- 0.013; mean +/- SD) as was muscle PO2, with modal values between 4 and 60 mmHg (0.5 and 8 kPa). CONCLUSIONS: Angiogenesis during normal muscle growth does not maintain CD, but with similar heterogeneity of capillary spacing it preserves the potential for adequate intramuscular oxygenation.     

Nail fold capillaroscopy findings in patients with primary fibromyalgia

Summary The nail fold capillary morphology and blood flow were examined by capillaroscopy in 10 patients with primary fibromyalgia. Only slight morphological anomalies such as moderate enlargement of capillary loops and variations in calibre were found. No obvious correlation emerged between capillary morphology and the duration of the disease, smoking, or history of Raynaud's phenomenon. Three patients with a history of Raynaud's phenomenon showed sluggish capillary flow correlated with subnormal skin temperature during registration. The findings suggest that marked generalized capillary abnormality such as that often involving the nail fold capillaries in many connective tissue disorders is not a prominent feature of primary fibromyalgia.     

Three-dimensional reconstruction of brain capillaries from frozen serial sections.

Based on frozen serial sections and microphotographs, the present study describes a three-dimensional reconstruction of the alkaline-phosphatase positive capillary network in the cat's cerebral cortex. This model clearly demonstrates a continuous increase of the volumetric capillary density towards the deeper cortical layers. Results of a morphometric investigation subsequently performed on photographed capillaries and obtained from a relatively small tissue sample fit well with the data from the whole temporo-parietal cortex region. The correspondence between the two data sets reflects specific morphometric characteristics of the capillary net in the cortex of the temporo-parietal Suprasylvian Gyrus. The information obtained from the three-dimensional capillary model represents a basic tool for additional computer-assisted steric reconstruction of the capillary network in the cerebral cortex.     

Remodeling of Capillary Network in Left Ventricular Subendocardial Tissues Induced by Intravenous Vasopressin Administration

Objective: The question of whether the coronary vasospasm induced by intravenous administration of vasopressin produces any remodeling of the capillary network in the left ventricle was investigated. To this end, cardiac tissues obtained from vasopressin-injected rats were stained to allow capillary counting and for basic fibroblast growth factor (bFGF). Methods: Nine male Donryu rats were divided into three groups that received, respectively, 0.25 ml of saline containing 0, 0.5, or 1.0 U/kg vasopressin injected into the tail vein once daily for 4 days. Rats were killed 30 days after the last injection. Two additional rats each received a single intravenous injection of 1.0 U/kg vasopressin and were killed 24 hours later. The left ventricles were removed and 16- or 10-μm frozen sections were cut for differential staining and distribution of bFGF, respectively. Differential staining was used to classify the capillary portions, and bFGF was identified by immunohistological staining. Results: Compared with the control group, total capillary density was increased in both vasopressin-treated groups, capillary to myocyte ratio was increased, and the capillary domain areas decreased in the three capillary portions. Arteriolar and intermediate capillary portions increased, while the venular capillary portion decreased. In rats killed 24 hours after vasopressin injection, a considerable amount of bFGF could be demonstrated immunohistochemically in the ventricular tissues, and the punctate distribution of bFGF was still found in rats killed 30 days after treatment. Conclusions: A remodeling of capillary network which would increase the oxygen transport capacity to cardiac tissues was produced in left ventricular tissues by intravenous injection of vasopressin. bFGF located around capillaries and in the interstitial space may have been involved in the capillary remodeling.     

Capillary geometrical changes with fiber shortening in rat myocardium.

Capillary-to-fiber geometrical relations constitute an integral component of peripheral gas exchange. Determination of capillary length and surface area density necessitates quantification of capillary orientation (i.e., tortuosity and branching). In skeletal muscle, capillary tortuosity increases in a curvilinear fashion at reduced sarcomere length, and this compensates for decreased capillary density as fiber cross-sectional area increases. To investigate these relations in myocardium, rat hearts were glutaraldehyde perfusion-fixed in calcium- or barium-induced "systole" to provide varying degrees of fiber shortening. Morphometric techniques were used to analyze capillary geometry in subepicardium (EPI) and subendocardium (ENDO) using 1-micron sections cut transverse and longitudinal to the muscle fiber axis. Capillary density on transverse and longitudinal sections, capillary diameter, fiber cross-sectional area, and sarcomere length were determined in each region. Capillary surface density was computed, and values were related to sarcomere length and compared with published data for diastolic hearts. Sarcomere length in systole ranged from 2.06 +/- 0.03 to 1.35 +/- 0.02 microns (EPI) and from 1.93 +/- 0.04 to 1.44 +/- 0.04 microns (ENDO). Fiber cross-sectional area (EPI, 344 +/- 13 microns2; ENDO, 343 +/- 12 microns2) was significantly larger, and capillary density on transverse sections was significantly smaller (EPI, 4,105 +/- 318 mm-2; ENDO, 4,145 +/- 267 mm-2) than in hearts arrested in diastole. Compared with skeletal muscle, capillary tortuosity was substantially less increased by fiber shortening. Capillary tortuosity and branching did not differ between EPI and ENDO and contributed a maximum of 33% (range, 13-33%) to capillary length density and surface area at a sarcomere length of 1.45 +/- 0.04 microns. Compared with diastolic hearts, capillary length density decreased on average by 19.6% (EPI) and 17.7% (ENDO); similarly, capillary surface density decreased 19.9% (EPI) and 13.7% (ENDO). We conclude that, with fiber shortening in the heart, fiber cross-sectional area increases and capillary numerical density decreases as predicted from reduced sarcomere length. Combined with the minimal geometrical changes of the capillary bed at shorter fiber lengths, this results in a lower capillary length and surface area per fiber volume in systole. Consequently, the structural potential for O2 diffusion into myocytes is determined, in part, by fiber length.     

L-arginine and antineutrophil serum enable venular control of capillary perfusion in hypercholesterolemic rats

OBJECTIVE: The purpose of this study was to investigate and counteract dysfunctional control of capillary flow in hypercholesterolemia. Capillary flow is controlled by arteriolar tone, which in turn is influenced by mediators released from closely paired venules in a mechanism that involves nitric oxide (NO). However, venular control of capillary flow is altered with hypercholesterolemia. METHODS: Rats were given a normal or high-cholesterol diet before measurements of mesenteric capillary red blood cell velocity. The arteriolar pathway leading to the capillary was videotaped to measure the percent of the surrounding area (within 15 |gmm) that was occupied by a venule (% pairing). RESULTS: Venule-paired arterioles were significantly smaller in hypercholesterolemia compared with normocholesterolemia, corresponding to slower capillary flow. A positive correlation between capillary velocity and % pairing observed in normocholesterolemia was not observed during NO synthase inhibition or in hypercholesterolemic rats. However, positive correlations between the two parameters were found in hypercholesterolemia when the rats were given drinking water supplementation of L-arginine or an injection of antineutrophil serum, both of which tended to improve velocity in capillaries branching from venule-paired arteriolar pathways. CONCLUSIONS: Dysfunctional venular control of capillary perfusion in hypercholesterolemia may be a consequence of a neutrophil-mediated deficiency of NO.     

Effects of aging on capillary number and luminal size in rat soleus and plantaris muscles

To clarify aging-related changes in the capillary network in skeletal muscle, we morphometrically examined the capillary supply to individual muscle fibers and capillary luminal size in young (3-month-old) and old (19-month-old) male Wistar rats. All morphometric parameters for capillary and muscle fiber were determined in the cross sections of the perfusion-fixed soleus (SOL) and plantaris (PL) muscles. The range of fiber size was larger in the old muscles because of hypertrophy and atrophy of fibers. However, the capillary supply to individual muscle fibers, assessed as the mean of capillary contacts around a muscle fiber, did not change with aging in SOL muscle (young rats = 7.8 +/- 0.4 vs old rats = 8.1 +/- 0.8) or PL muscle (young rats = 6.4 +/- 0.3 vs old rats = 7.0 +/- 0.9). The ratio of individual muscle fiber area to the number of capillary contacts around a muscle fiber did not differ between young rats (SOL = 361.7 +/- 76.0; PL = 264.7 +/- 20.9) and old rats (SOL = 350.2 +/- 61.3; PL = 296.8 +/- 44.9). The mean capillary luminal diameter did not differ statistically in young and old rats (SOL, young rats = 5.3 +/- 0.5 vs old rats = 5.1 +/- 0.1; PL, young rats = 5.0 +/- 0.3 vs old rats = 5.4 +/- 0.2). In conclusion, the relationship between capillary supply and muscle fiber size is similar for both young and old rats, and the luminal size of each capillary was maintained with advancing age.     

Quantitative studies in the cerebral cortex of aging humans

The capillary network of the human cerebral cortex was morphometrically investigated in a young, aged and aged hypertonic group. In the aging cortex, augmented values of capillary diameter and volume are accompanied by smaller distances between capillaries and an extended length per unit cortex volume. An increased capillary diameter of the aged hypertonic group is probably due to a high blood pressure. The regulating influence of the surrounding astroglia and the extracellular fluid on capillaries seems to lack. In comparison to normotonic brains, higher distances between capillaries and a decreased capillary length were measured.