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Blood perfusion tubing . The schematic shows the tubing setup used for circulating blood through the isolated lung preparation. Also indicated are associated components through which the tubing is routed. A schematic of the heart and lung is included to show sites of connection between the tubing and pulmonary artery ( PA ), and left atrial ( LA ) cannula (blue dashed lines).
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The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. T...
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... Isolated blood-perfused lungs were prepared from rats and mice, as reported. 14,15 Briefly, animals were anesthetized by intraperitoneal injection of ketamine and xylazine. Heparin was injected via cardiac puncture, and the animals were exsanguinated. ...
Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca(2+) oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messenger different from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelial connexin43, suggesting a role for connexin43 gap junctions. Compartmental second messenger signaling and interendothelial communication define the specific second messenger involved in exacerbating proinflammatory responses to receptor-mediated agonists.
... Briefly, a PE10 (BD Biosciences, Sparks, MD) microcatheter was introduced through the left atrial cannula, and blood cell-free conditions were established by flushing with HEPES-buffered Ringer's solution into microvessels in a small portion of the lung. A video of the procedure along with photographs showing the final size of the blood-free region was reported recently (15). To quantify microvessel permeability, the fluorescent tracer FDx20 was infused into the cleared microvessels, and the FDx20 fluorescence was captured at regular intervals (1 image/min) using a wide-angle microscope (Olympus BX61-WI). ...
Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. As inhibiting connexin43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations post-instillation, the lungs were isolated and blood-perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that Cx43 expression and microvessel permeability, both increased above baseline within a few hours after endotoxin instillation, but declined progressively over the next few days. At day 5 post-endotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of inter-microvessel communication determined by photolytic uncaging of Ca(2+). However by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline, and then returning to baseline at longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin and microvessel permeability, with changes in Cx43 temporally leading the other responses.
The lung represents a unique immune environment. The primary function of the lung is to enable gas exchange by facilitating the transfer of oxygen into and carbon dioxide out of the blood. However, as a direct byproduct of this process the lung is also constantly exposed to particles, allergens, and pathogens alongside air itself. Due to this, the pulmonary immune system exists in a fine balance between quiescence and inflammation, deviations from which can lead to a failure in respiratory function. A rich history exists attempting to define the critical features of lung immunity, and most recently advances in intravital microscopy have enabled the visualization of intercellular immune dynamics in both steady-state and a variety of disease conditions. In this review, we will summarize a variety of approaches to intravital lung imaging as well as how its application has advanced our understanding of normal lung function as well as disease states such as pulmonary metastasis, asthma, and lung injury.
Pulmonary blood vessels act as a well-regulated barrier to the flux of fluid and solutes between the lumen and the air space. Perturbation of the barrier function results in excessive fluid leak into the interstitium and alveoli, and impairs gas exchange. Recent studies provide deeper insight into the precise control mechanisms involved in the regulation of the barrier. This chapter will highlight these mechanisms and discuss the current understanding on the fluid and solute transport pathways across the vascular endothelial layer. In addition, the chapter summarizes the contributions of extra-endothelial structures such as pericytes and glycocalyx in regulating fluid flux across pulmonary vessels. The chapter concludes with an analysis on the impact of pulmonary endothelial heterogeneity and experimental models on current interpretations of barrier function and regulatory mechanisms.
Much recent interest in lung bioengineering by pulmonary investigators, industry and the organ transplant field has seen a rapid growth of bioreactor development ranging from the microfluidic scale to the human-sized whole lung systems. A comprehension of the findings from these models is needed to provide the basis for further bioreactor development.
The goal was to comprehensively review the current state of bioreactor development for the lung.
A search using PubMed was done for published, peer-reviewed papers using the keywords "lung" AND "bioreactor" or "bioengineering" or "tissue engineering" or "ex vivo perfusion".
Many new bioreactors ranging from the microfluidic scale to the human-sized whole lung systems have been developed by both academic and commercial entities. Microfluidic, lung-mimic and lung slice cultures have the advantages of cost-efficiency and high throughput analyses ideal for pharmaceutical and toxicity studies. Perfused/ventilated rodent whole lung systems can be adapted for mid-throughput studies of lung stem/progenitor cell development, cell behavior, understanding and treating lung injury and for preliminary work that can be translated to human lung bioengineering. Human-sized ex vivo whole lung bioreactors incorporating perfusion and ventilation are amenable to automation and have been used for whole lung decellularization and recellularization. Clinical scale ex vivo lung perfusion systems have been developed for lung preservation and reconditioning and are currently being evaluated in clinical trials.
Significant advances in bioreactors for lung engineering have been made at both the microfluidic and the macro scale. The most advanced are closed systems that incorporate pressure-controlled perfusion and ventilation and are amenable to automation. Ex vivo lung perfusion systems have advanced to clinical trials for lung preservation and reconditioning. The biggest challenges that lie ahead for lung bioengineering can only be overcome by future advances in technology that solve the problems of cell production and tissue incorporation.
