Optimizing imaging of the rat pulmonary microvasculature by micro-computed tomography

Micro-computed tomography (micro-CT) is used in pre-clinical research to generate high-resolution three-dimensional (3D) images of organs and tissues. When combined with intravascular contrast agents, micro-CT can provide 3D visualization and quantification of vascular networks in many different organs. However, the lungs present a particular challenge for contrast perfusion due to the complexity and fragile nature of the lung microcirculation. The protocol described here has been optimized to achieve consistent lung perfusion of the microvasculature to vessels < 20 microns in both normal and pulmonary arterial hypertension rats. High-resolution 3D micro-CT imaging can be used to better visualize changes in 3D architecture of the lung microcirculation in pulmonary vascular disease and to assess the impact of therapeutic strategies on microvascular structure in animal models of pulmonary arterial hypertension.

Effects of high PEEP and fluid administration on systemic circulation, pulmonary microcirculation, and alveoli in a canine model

This study aimed to determine the response of systemic circulation, pulmonary microcirculation, and alveoli to high positive end-expiratory pressure (PEEP) in a canine model. This study was conducted in nine mixed-breed dogs on mechanical ventilation under anesthesia. The PEEP was initially set at 5 cmH2O (PEEP5), the PEEP was then increased to 25 cmH2O (PEEP25), and then saline was used for fluid loading. Data were obtained at the following time points: PEEP5; PEEP25 prefluid loading; and PEEP25 postfluid loading. The images of subpleural lung microcirculation were assessed by sidestream dark-field microscopy, and the hemodynamic data were collected from pulse contour waveform-derived measurements. Compared with PEEP5, the lung microvascular flow index (MFI, 2.3 ± 0.8 versus 0.9 ± 0.8, P = 0.001), lung perfused vessel density (PVD, 4.2 ± 2 versus 1.5 ± 1.8, P = 0.004), lung proportion of perfused vessel (PPV, 93 ± 14 versus 40 ± 4, P = 0.003), cardiac output (2.5 ± 0.6 versus 1.4 ± 0.5, P = 0.001), and mean blood pressure (116 ± 24 versus 91 ± 31, P = 0.012) were significantly lower at PEEP25 prefluid loading. After fluid loading, there were no significant differences in cardiac output or mean arterial pressure between the PEEP5 and PEEP25 postfluid loading levels. However, the lung microcirculatory MFI, PVD, and PPV at PEEP25 postfluid loading remain lower than at PEEP5. A significant increase in septal thickness was found at PEEP25 postfluid loading relative to septal thickness at PEEP25 prefluid loading (25.98 ± 5.31 versus 40.76 ± 7.9, P = 0.001). Under high PEEP, systemic circulation was restored after fluid loading, but lung microcirculation was not. Moreover, the septal thickness of alveoli significantly increased after fluid loading. NEW & NOTEWORTHY An excessively high positive end-expiratory pressure (PEEP) can impair the systemic circulation and alveolar microcirculation. In the high-PEEP condition, fluid loading restored the systemic circulation but did not affect the impaired lung microcirculation. The septal thickness of the alveoli significantly increased after fluid loading in the high-PEEP condition.

Understanding Vasomotion of Lung Microcirculation by In Vivo Imaging

The balance of lung extravascular water depends upon the control of blood flow in the alveolar distribution vessels that feed downstream two districts placed in parallel, the corner vessels and the alveolar septal network. The occurrence of an edemagenic condition appears critical as an increase in extravascular water endangers the thinness of the air–blood barrier, thus negatively affecting the diffusive capacity of the lung. We exposed anesthetized rabbits to an edemagenic factor (12% hypoxia) for 120 min and followed by in vivo imaging the micro-vascular morphology through a “pleural window” using a stereo microscope at a magnification of 15× (resolution of 7.2 μm). We measured the change in diameter of distribution vessels (50–200 μm) and corner vessels (<50 μm). On average, hypoxia caused a significant decrease in diameter of both smaller distribution vessels (about ~50%) and corner vessels (about ~25%) at 30 min. After 120 min, reperfusion occurred. Regional differences in perivascular interstitial volume were observed and could be correlated with differences in blood flow control. To understand such difference, we modelled imaged alveolar capillary units, obtained by Voronoi method, integrating microvascular pressure parameters with capillary filtration. Results of the analysis suggested that at 120 min, alveolar blood flow was diverted to the corner vessels in larger alveoli, which were found also to undergo a greater filtration indicating greater proneness to develop lung edema.

A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) – Part II: Enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis

SummaryWe have discovered a novel small-molecule TAFIa inhibitor, BX 528, which is potent, highly selective against other carboxypeptidases and safe. The present study was to determine if BX 528 can enhance exogenous and endogenous thrombolysis in four different animal models. In the first three models, a thrombus was induced by FeCl2 (dogs) or laser (rats) injury of the femoral artery, or formed ex vivo and implanted in the jugular vein in rabbits. A low dose of exogenous t-PA was given to induce a lowlevel thrombolysis on an established thrombus. Co-treatment with BX 528 further enhanced the thrombolytic effects induced by the exogenous t-PA and, thus, r educed thrombosis in all three animal models. In a second rat model, fibrin deposition in the lungs was induced by batroxobin, which was spontaneously resolved in 30 minutes due to the activation of endogenous fibrinolysis. Pre-treatment with lipopolysaccharide (LPS) attenuated this spontaneous fibrinolysis. Co-treatment with 10 mg/kg BX 528 prevented the LPS-induced attenuation of endogenous fibrinolysis. Thus, these studies demonstrated that inhibition ofTAFIa by BX 528, our newly discovered small-molecule TAFIa inhibitor, enhanced both the exogenous (induced by a low dose of t-PA) and endogenous (LPS-induced resistance) thrombolysis without increasing the bleeding risk in four different animal models of thrombosis in different species (rat, dog and rabbit) employing different thrombogenic stimuli (FeCl2, laser, ex vivo and batroxobin) to induce thrombus formation in different tissues (artery, vein and lung microcirculation).

The Phosphatidylserine Receptor-Phosphatidylserine Axis Promotes Sickle Erythrocyte Retention in the Lung Microcirculation.

Pulmonary vascular occlusion is a major cause of morbidity and mortality in sickle cell disease. Adhesion between sickle erythrocytes, leukocytes and the endothelium are intimately involved in pulmonary vascular occlusion, which contributes to the pathogenesis of the acute chest syndrome. Despite this significance, specific molecules and cognate mechanisms responsible for trapping sickle erythrocytes in the lung microcirculation are poorly understood. In this study, we show for the first time that products released by activated neutrophils act through the phosphatidyserine and phosphatidylserine receptor axis to promote retention of sickle erythrocytes in the lung microcirculation. We demonstrated that co-incubation with autologous activated neutrophils increased 2-fold the proportion of phosphatidylserine exposed sickle erythrocytes. This effect was abrogated by prior treatment of neutrophils with zileuton a pharmacological inhibitor of 5-lipoxygenase. We next determined that primary endothelial cells derived from the pulmonary vasculature expressed the stereospecific receptor for phosphatidylserine. Transcripts for phosphatidylserine receptor (PSR) were 2-fold more abundant in endothelial cells from the pulmonary microvasculature compared to those from the pulmonary artery. To determine the relevance of neutrophil activation in PSR expression, cultures of PMVECs were treated with supernatants of activated neutrophils from patients with sickle cell anemia, and this resulted in 2-fold increase in PSR transcripts. To unravel the pathophysiological relevance of these findings, isolated rat lungs were perfused with 51Cr labeled sickle erythrocytes pre-incubated with annexin V, and retention of sickle erythrocytes quantified by 51Cr radioactivity. The number of sickle erythrocytes trapped in isolated lungs was 2-fold lower compared to the value in control experiments performed with untreated sickle erythrocytes. In agreement with our previous findings activated neutrophils significantly increased sickle erythrocyte retention in the lung (P=0.0004), however cloaking of sickle erythrocytes with annexin V reduced retention by 3-fold. Collectively, these data show that activation of neutrophils increases expression of PSR and its cognate ligand on endothelial cells and sickle erythrocytes respectively, providing the molecular basis for an adhesion complex that increases retention of sickle erythrocytes in the pulmonary endothelium. The PSR-phosphatidylserine adhesion complex therefore offers a novel therapeutic target to reduce pulmonary vascular occlusive events in sickle cell disease.

Influence of lung volume on pulmonary microvascular pressure-volume characteristics

The pressure-volume (P-V) characteristics of the lung microcirculation are important determinants of the pattern of pulmonary perfusion and of red and white cell transit times. Using diffuse light scattering, we measured capillary P-V loops in seven excised perfused dog lobes at four lung volumes, from functional residual capacity (FRC) to total lung capacity (TLC), over a wide range of vascular transmural pressures (Ptm). At Ptm 5 cmH2O, specific compliance of the microvasculature was 8.6%/cmH2O near FRC, decreasing to 2.7%/cmH2O as lung volume increased to TLC. At low lung volumes, the vasculature showed signs of strain stiffening (specific compliance fell as Ptm rose), but stiffening decreased as lung volume increased and was essentially absent at TLC. The P-V loops were smooth without sharp transitions, consistent with vascular distension as the primary mode of changes in vascular volume with changes in Ptm. Hysteresis was small (0.013) at all lung volumes, suggesting that, although surface tension may set basal capillary shape, it does not strongly affect capillary compliance.