Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.     

The Reciprocity of M- and N-Cholinergic Mechanisms in the Pulmonary Microcirculatory Changes in Case of Experimental Pulmonary Embolism

Journal of Evolutionary Biochemistry and Physiology - In acute experiments on isolated perfused rabbit’s lungs, the changes of pulmonary microhemodynamics were studied when modeling pulmonary...     

红腹锦鸡肺的组织结构与微血管构筑

为了了解红腹锦鸡(Chroysolophus pictus)肺的微细结构和微血管构筑特征,为呼吸生物学研究提供形态学依据,用组织学方法和微血管铸型技术在光镜和扫描电镜下观察研究了红腹锦鸡肺的组织结构与微血管构筑情况。结果表明,红腹锦鸡肺主要由各级支气管构成,从三级支气管上呈辅射状分出许多呼吸毛细管(微气管),并相互吻合成网状,呼吸毛细管外面包围有丰富的毛细血管;红腹锦鸡肺毛细血管垂直围绕在各微气管外,并相互吻合成密集的立体微血管网;毛细血管管径4.5~7.0μm,微气管直径11~50μm。并对肺微血管构筑情况与呼吸效率的关系作了探讨。     

Determining Microvascular Obstruction and Infarct Size with Steady-State Free Precession Imaging Cardiac MRI

Purpose

In cardiac MRI (cMRI) injection of contrast medium may be performed prior to the acquisition of cine steady-state free precession (SSFP) imaging to speed up the protocol and avoid delay before late Gadolinium enhancement (LGE) imaging. Aim of this study was to evaluate whether a condensed clinical protocol with contrast cine SSFP imaging is able to detect early microvascular obstruction (MO) and determine the infarct size compared to the findings of LGE inversion recovery sequences.

Materials and Methods

The study complies with the Declaration of Helsinki and was performed following approval by the ethic committee of the University of Erlangen-Nuremberg. Written informed consent was obtained from every patient. 68 consecutive patients (14 females/54 males) with acute ST-elevation myocardial infarction (STEMI) treated by percutaneous coronary revascularization were included in this study. CMRI was performed 6.6±2 days after symptom onset and MO and infarct size in early contrast SSFP cine imaging were compared to LGE imaging.

Results

MO was detected in 47/68 (69%) patients on cine SSFP and in 41/68 (60%) patients on LGE imaging. In 6 patients MO was found on cine SSFP imaging but was not detectable on LGE imaging. Infarct size on cine SSFP showed a strong agreement to LGE imaging (intraclass correlation coefficient [ICC] of 0.96 for enddiastolic, p<0.001 and 0.96 for endsystolic, p<0.001 respectively). Significant interobserver agreement was found measuring enddiastolic and endsystolic infarct size on cine SSFP imaging (p<0.01).

Conclusions

In patients after STEMI infarct size and presence of MO can be detected with contrast cine SSFP imaging. This could be an option in patients who are limited in their ability to comply with the demands of a cMRI protocol.     

Effects of C-peptide on Microvascular Blood Flow and Blood Hemorheology

Beside functional and structural changes in vascular biology, alterations in the rheologic properties of blood cells mainly determines to an impaired microvascular blood flow in patients suffering from diabetes mellitus. Recent investigations provide increasing evidence that impaired C-peptide secretion in type 1 diabetic patients might contribute to the development of microvascular complications. C-peptide has been shown to stimulate endothelial NO secretion by activation of the Ca²⁺ calmodolin regulated enzyme eNOS. NO himself has the potency to increase cGMP levels in smooth muscle cells and to activate Na⁺ K⁺ ATPase activity and therefore evolves numerous effects in microvascular regulation. In type 1 diabetic patients, supplementation of C-peptide was shown to improve endothelium dependent vasodilatation in an NO-dependent pathway in different vascular compartments. In addition, it could be shown that C-peptide administration in type 1 diabetic patients, results in a redistribution of skin blood flow by increasing nutritive capillary blood flow in favour to subpapillary blood flow. Impaired Na⁺ K⁺ ATPase in another feature of diabetes mellitus in many cell types and is believed to be a pivotal regulator of various cell functions. C-peptide supplementation has been shown to restore Na⁺ K⁺ATPase activity in different cell types during in vitro and in vivo investigations. In type 1 diabetic patients, C-peptide supplementation was shown to increase erythrocyte Na⁺ K⁺ATPase activity by about 100%. There was found a linear relationship between plasma C-peptide levels and erythrocyte Na⁺ K⁺ATPase activity. In small capillaries, microvascular blood flow is increasingly determined by the rheologic properties of erythrocytes. Using laser-diffractoscopie a huge improvement in erythrocyte deformability could be observed after C-peptide administration in erythrocytes of type 1 diabetic patients. Inhibition of the Na⁺ K⁺ATPase by Obain completely abolished the effect of C-peptide on erythrocyte deformability. In conclusion, C-peptide improves microvascular function and blood flow in type 1 diabetic patients by interfering with vascular and rheological components of microvascular blood flow.     

Measurement of Cutaneous Microvascular Exudates Using Evans Blue

As an approach to the study of inflammation, strategies were evaluated for quantitative assessment of plasma exudate from thecutaneous microvasculature. Measurements were based on recovery of Evans blue dye (EB) from rat skin. After preliminary studies to evaluate extraction methods, almost complete EB recovery was accomplished by homogenizing tissue in a mixture of acetone, water and sodium sulfate. When sources of potential variation were identified, expression of results as agonist-induced plasma accumulation provided precise results based on EB measurements. Also. the feasibility of parallel biochemical studies was demonstrated.     

Development of a Real-Time Resistance Measurement for Vibrio parahaemolyticus Detection by the Lecithin-Dependent Hemolysin Gene

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg²⁺), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg²⁺) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1–2 hours, with a minimum bacterial density of 10 CFU.mL⁻¹ and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.     

Measurement of Cutaneous Microvascular Exudates Using Evans Blue

As an approach to the study of inflammation, strategies were evaluated for quantitative assessment of plasma exudate from thecutaneous microvasculature. Measurements were based on recovery of Evans blue dye (EB) from rat skin. After preliminary studies to evaluate extraction methods, almost complete EB recovery was accomplished by homogenizing tissue in a mixture of acetone, water and sodium sulfate. When sources of potential variation were identified, expression of results as agonist-induced plasma accumulation provided precise results based on EB measurements. Also. the feasibility of parallel biochemical studies was demonstrated.     

钙离子浓度动态变化

目的:探讨应用激光共聚焦扫描显微镜(LSCM)技术检测缺氧状态的人肺微血管内皮细胞(HPMVEC)内钙离子(Ca2+)浓度动态变化的价值。方法:HPMVEC常规培养,按观察时间点不同分为5个缺氧培养组(1h hyp组、2h hyp组、4h hyp组、6h hyp组和8h hyp组)以及1个对照组(0h con组)共6个组,每组设8个复孔,应用LSCM技术测定缺氧后HPMVEC内Ca2+浓度水平及随时间推移的变化。结果:LSCM技术显示HPMVEC内Ca2+的荧光强度1h hyp组与0h con组比较、2h hyp组与1h hyp组比较、4h hyp组与2h hyp组比较、6h hyp组与4h hyp组比较、8h hyp组与6h hyp组比较有显著差异(P<0.05)。线性回归分析结果显示Ca2+荧光强度与缺氧时间成正相关(r=0.969,P<0.01)。结论:HPMVEC内Ca2+浓度随缺氧时间增长而增高;LSCM在动态检测缺氧状态下HPMVEC内的Ca2+浓度变化中具有明显优势。     

应用激光共聚焦扫描显微镜技术检测缺氧人肺微血管内皮细胞内钙离子浓度动态变化

目的：探讨应用激光共聚焦扫描显微镜（LSCM）技术检测缺氧状态的人肺微血管内皮细胞（HPMVEC）内钙离子（Ca2＋）浓度动态变化的价值。方法：HPMVEC常规培养,按观察时间点不同分为5个缺氧培养组（1h hyp组、2h hyp组、4h hyp组、6h hyp组和8h hyp组）以及1个对照组（0h con组）共6个组,每组设8个复孔,应用LSCM技术测定缺氧后HPMVEC内Ca2＋浓度水平及随时间推移的变化。结果：LSCM技术显示HPMVEC内Ca2＋的荧光强度1h hyp组与0h con组比较、2h hyp组与1h hyp组比较、4h hyp组与2h hyp组比较、6h hyp组与4h hyp组比较、8h hyp组与6h hyp组比较有显著差异（P〈0.05）。线性回归分析结果显示Ca2＋荧光强度与缺氧时间成正相关（r=0.969,P〈0.01）。结论：HPMVEC内Ca2＋浓度随缺氧时间增长而增高;LSCM在动态检测缺氧状态下HPMVEC内的Ca2＋浓度变化中具有明显优势。     

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca²⁺ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)^1-3 and hyperpolarization^4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca²⁺ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions^6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.     

Numerical Simulation of Blood Flow Through Microvascular Capillary Networks

A numerical method is implemented for computing blood flow through a branching microvascular capillary network. The simulations follow the motion of individual red blood cells as they enter the network from an arterial entrance point with a specified tube hematocrit, while simultaneously updating the nodal capillary pressures. Poiseuille’s law is used to describe flow in the capillary segments with an effective viscosity that depends on the number of cells residing inside each segment. The relative apparent viscosity is available from previous computational studies of individual red blood cell motion. Simulations are performed for a tree-like capillary network consisting of bifurcating segments. The results reveal that the probability of directional cell motion at a bifurcation (phase separation) may have an important effect on the statistical measures of the cell residence time and scattering of the tube hematocrit across the network. Blood cells act as regulators of the flow rate through the network branches by increasing the effective viscosity when the flow rate is high and decreasing the effective viscosity when the flow rate is low. Comparison with simulations based on conventional models of blood flow regarded as a continuum indicates that the latter underestimates the variance of the hematocrit across the vascular tree.     

Simple Modification of the Bladder Outlet Obstruction Index for Better Prediction of Endoscopically-Proven Prostatic Obstruction: A Preliminary Study

Purpose

The bladder outlet obstruction index (BOOI), also known as the Abrams-Griffiths (AG) number, is the most widely used index for predicting BOO. However, the obstructed prostatic urethra determined by the BOOI is often inconsistent with endoscopically-proven obstruction. We assessed abdominal straining pattern as a novel parameter for improving the prediction of BOO.

Materials and Methods

We retrospectively reviewed the pressure-flow studies (PFS) and cystourethroscopy in 176 BPH/LUTS patients who were unresponsive to medical therapy. During PFS, some groups of patients tried to urinate with abdominal straining, which can increases intravesical pressure and underestimate BOOI theoretically. Accordingly, the modified BOOI was defined as (P_detQ_max+ΔP_abd)-2Q_max.

Results

Ultimately, 130 patients were eligible for the analysis. In PFS, ΔP_abd (P_abdQ_max-initial P_abd) was 11.81±13.04 cmH₂O, and it was 0–9 cmH₂O in 75 (57.7%), 10–19 cmH₂O in 23 (17.7%) and ≥20 cmH₂O in 32 (24.6%) patients. An endoscopically obstructed prostatic urethra in 92 patients was correctly determined in 47 patients (51.1%) by the original BOOI versus 72 patients (78.3%) based on the modified BOOI. Meanwhile, an “unobstructed” urethra according to the original BOOI was present in 11 patients (12.0%), whereas according to the modified BOOI, only 2 (2.1%) would be labeled as “unobstructed”. In receiver operating characteristic curves, the area under the curve was 0.906 using the modified BOOI number versus 0.849 in the original BOOI (p<0.05).

Conclusions

The change in abdominal pressure was correlated with endoscopically-proven obstruction. Our simple modification of the BOOI on the basis of this finding better predicted bladder outlet obstruction and, therefore, should be considered when evaluating BOO in patients with LUTS/BPH.     

The Effects of Time-Varying Blood Flow on Diffusional Resistance to Oxygen Transfer in the Pulmonary Capillaries

The effects of the persistence of pulsatile blood flow in the pulmonary capillaries on the over-all diffusing capacity and alveolar-arterial oxygen tension gradient were studied. A mathematical analysis was made of the oxygen transfer process using an undamped cardiac flow pulse in the capillaries and taking into account the finite rate of reaction of oxygen with hemoglobin.

In five cases of both normal and low oxygen atmospheres, combined with varying degree of exercise, it was found that the alveolar-arterial oxygen tension gradients were not affected by the time-varying blood flow, while in cases of breathing air the over-all diffusing capacity of the lung increased 10-15% over the diffusing capacity obtained with constant blood flow rate in the pulmonary capillaries.

     

Daily Peak Flow Measurements in the Assessment of Steroid Therapy for Airway Obstruction

Measurements of peak expiratory flow can be made by outpatients on themselves. This technique, which may be carried out at home, has proved to be valuable in assessing the effect of steroids and other bronchodilator drugs in relieving airway obstruction.     

A Combined Pulmonary Function and Emphysema Score Prognostic Index for Staging in Chronic Obstructive Pulmonary Disease

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is characterized by high morbidity and mortality. Lung computed tomography parameters, individually or as part of a composite index, may provide more prognostic information than pulmonary function tests alone.

Aim

To investigate the prognostic value of emphysema score and pulmonary artery measurements compared with lung function parameters in COPD and construct a prognostic index using a contingent staging approach.

Material-Methods

Predictors of mortality were assessed in COPD outpatients whose lung computed tomography, spirometry, lung volumes and gas transfer data were collected prospectively in a clinical database. Univariate and multivariate Cox proportional hazard analysis models with bootstrap techniques were used.

Results

169 patients were included (59.8% male, 61.1 years old; Forced Expiratory Volume in 1 second % predicted: 40.5±19.2). 20.1% died; mean survival was 115.4 months. Age (HR = 1.098, 95% Cl = 1.04–1.252) and emphysema score (HR = 1.034, 95% CI = 1.007–1.07) were the only independent predictors of mortality. Pulmonary artery dimensions were not associated with survival. An emphysema score of 55% was chosen as the optimal threshold and 30% and 65% as suboptimals. Where emphysema score was between 30% and 65% (intermediate risk) the optimal lung volume threshold, a functional residual capacity of 210% predicted, was applied. This contingent staging approach separated patients with an intermediate risk based on emphysema score alone into high risk (Functional Residual Capacity ≥210% predicted) or low risk (Functional Residual Capacity <210% predicted). This approach was more discriminatory for survival (HR = 3.123; 95% CI = 1.094–10.412) than either individual component alone.

Conclusion

Although to an extent limited by the small sample size, this preliminary study indicates that the composite Emphysema score-Functional Residual Capacity index might provide a better separation of high and low risk patients with COPD, than other individual predictors alone.