Fluorescent microangiography (FMA): an improved tool to visualize the pulmonary microvasculature

Abstract

Visualization of the complex lung microvasculature and resolution of its three-dimensional architecture remains a difficult experimental challenge. We present a novel fluorescent microscopy technique to visualize both the normal and diseased pulmonary microvasculature. Physiologically relevant pulmonary perfusion conditions were applied using a low-viscosity perfusate infused under continuous airway ventilation. Intensely fluorescent polystyrene microspheres, confined to the vascular space, were imaged through confocal optical sectioning of 200 lm-thick lung sections. We applied this technique to rat lungs and the markedly enhanced depth of field in projected images allowed us to follow vascular branching patterns in both normal lungs and lungs from animals with experimentally induced pulmonary arterial hypertension. In addition, this method allowed complementary immunostaining and identification of cellular components surrounding the blood vessels. Fluorescent microangiography is a widely applicable and quantitative tool for the study of vascular changes in animal models of pulmonary disease. Keywords: confocal microscopy; microvasculature; pulmonary hypertensionThe circulatory bed of the lung is uniquely designed to accommodate the entire cardiac output and thus, remodeling of the microvasculature can play a dominant role in the progression of many pulmonary pathologies. For example, pulmonary hypertension (PH) is characterized by a marked increase in vascular resistance, likely resulting from a loss of pulmonary microvessels and/or from excessive smooth muscle hyperplasia in arterioles that normally display only incomplete muscularization. 1 Therefore, obtaining accurate three-dimensional (3D) morphological information on arteriolar and capillary structure along with spatial colocalization of biological mediators will provide fundamental mechanistic insights into the pathogenesis of many pulmonary diseases. 2 Animal models have been instrumental in the study of pulmonary diseases and numerous rodent models have been established. First generation models have involved toxin-induced pulmonary injuries, 3,4 whereas second and third generation models have been developed with transgenic techniques in combination with environmental or other biological stimuli. 5,6 Such models are powerful tools, yet require specialized techniques in order to efficiently characterize the temporal and spatial patterns of pulmonary pathogenesis. Owing to the technical complexity of current techniques, indepth characterization of pulmonary microvasculature remodeling has not been performed in these rodent models. Consequently, in order for these models to reach their full potential, newer, readily available techniques need to be developed.In this study, we present a novel and reproducible method to evaluate the morphometry of the pulmonary circulation and colocalize relevant pathological changes in the surrounding tissue, on a microscopic

Subtraction micro-computed tomography of angiogenesis and osteogenesis during bone repair using synchrotron radiation with a novel contrast agent

“…Indeed, a similarly low-concentrated agarose solution containing barium or fluorescent microbeads showed easy vascular perfusability in earlier studies. 33,34 Furthermore, the hydrophilicity of ZrCA could contribute to militating for its vascular penetration. That is, ZrCA can be easily miscible with the pre-perfused buffer solution, thereby reducing a possibility of patchy perfusion 16 in a non-tree-like vascular network, where diffusion, rather than convection, may be the primary transport mechanism in some vessels.…”

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

“…Kidney microvascular architecture was assessed in CAST/Ei mice injected with PBS or Stx2 (2,400 pg/g) using a fluorescence microangiography technique, essentially as described previously (79). In brief, animals were anesthetized with inhaled isoflurane 2.5%.…”

Early Combined Treatment with Cilostazol and Bone Marrow-Derived Endothelial Progenitor Cells Markedly Attenuates Pulmonary Arterial Hypertension in Rats

“…The protocol for fluorescent microsphere preparation was modified from that reported previously (Dutly et al, 2006). In brief, low melting point agarose (100 mg) (Sigma-Aldrich) was first dissolved in 9 ml of distilled water at 80°C, followed by the addition of 1 ml of greenish yellow fluorescent polystyrene 0.2-m microspheres (505-nm excitation, 515-nm emission) (FluoSpheres; Invitrogen) into the agarose solution.…”