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. 2014 Jul 7;3(2):e29748.
doi: 10.4161/intv.29748.

Quantitative Intravital Two-Photon Excitation Microscopy Reveals Absence of Pulmonary Vaso-Occlusion in Unchallenged Sickle Cell Disease Mice

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Quantitative Intravital Two-Photon Excitation Microscopy Reveals Absence of Pulmonary Vaso-Occlusion in Unchallenged Sickle Cell Disease Mice

Margaret F Bennewitz et al. Intravital. .
Free PMC article

Abstract

Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus.

Keywords: Two-photon microscopy; acute chest syndrome; neutrophils; red blood cells; sickle cell disease; vaso-occlusion.

Figures

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Figure 1. Comparison of single photon excitation (SPE) with two-photon excitation (TPE). (A) A single photon of higher energy (E = hc/λ1) is absorbed in SPE to produce fluorescence emission. In TPE, two lower energy photons (each with E = hc/λ2) are absorbed nearly simultaneously to produce the same fluorescence effect. Near-infrared lasers are used in TPE, which emit photons that have double the wavelength of photons used in SPE (λ2 = 2λ1). hc/λ is the energy of a photon, h is Planck’s constant, c is the speed of light, and λ is wavelength. (B) In SPE techniques, such as confocal microscopy, a cone of fluorescent light is emitted within the sample. To obtain an image localized to the focal plane, a pinhole must be used to filter the out-of-focus light in SPE. In TPE, fluorescence is inherently localized to the focal plane and thus all emitted photons can be collected. The localized fluorescence in TPE leads to less phototoxicity and less photobleaching as compared with SPE.
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Figure 2. Schematic of our TPE imaging setup. A catheter is placed into the carotid artery to enable iv delivery of intravascular fluorochromes and fluorescent antibodies. The mouse is intubated to facilitate mechanical ventilation and delivery of 1% isoflurane with FiO2 of 0.95. The temperature of the mouse is maintained with a heated stage and a temperature controlled enclosure surrounding the TPE microscope stage. Gentle vacuum suction is applied to the thoracic window device to immobilize a small region of the left lobe of the lung against a cover glass. TPE imaging is performed with a NIR laser. In the objective, red denotes excitation and green denotes emission fluorescence. The carotid artery catheter is also connected to a pressure transducer to monitor blood pressure during imaging. Pulse oximetery is used to monitor blood oxygen saturation, breath rate, breath distension, heart rate, and pulse distention.
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Figure 3. Vacuum enabled thoracic window allows for stable visualization of the pulmonary microcirculation and alveoli in a live C57BL/6 mouse. The pulmonary capillaries surrounding the alveoli (*) can be seen moving in and out of the imaging plane in the z-direction due to the dynamic expansion and contraction of the alveoli (*) with mechanical ventilation. Movement of alveoli starts from the bottom of the image at t = 0 s and moves up to the top with increasing time. Alveoli are marked by asterisks. Intravascular FITC dextran highlights the pulmonary capillaries and a feeding arteriole in green. The open arrow denotes the direction of blood flow within the feeding arteriole. The times displayed are relative to the selected video frames. Scale bars are 20 µm. The feeding arteriole has a diameter of 33 µm, while the capillaries have an average diameter of 6 ± 2 µm. The complete video sequence is included in Movie S1.
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Figure 4. Elongated neutrophils slowly transit or rapidly ‘hop’ through the pulmonary capillaries of a live C57BL/6 mouse. (A) Green channel. Neutrophils (green) were stained by intravascular (iv) injection of FITC Ly-6G mAb. Solid arrow denotes slowly transiting neutrophils. The dotted circle highlights a neutrophil that travels much more rapidly and can be seen hopping between video frames. (B) Red channel. Texas Red-conjugated dextran (red) was administered iv to visualize pulmonary capillaries and a feeding arteriole. The open arrow shows the direction of blood flow within the feeding arteriole. (C) Merged dual color image. Neutrophils (green) are shown trafficking through pulmonary capillaries (red). Neutrophils take on an elongated shape that fills the lumen of pulmonary capillaries. (D) Individual tracks of transiting neutrophils tracked over a 5 min observation period using Imaris software are shown. The tracking video was analyzed in Imaris to extract mean track speed and track length for all the neutrophils and is plotted as cumulative distribution graphs. Note that greater than 90% of neutrophils migrate at 0.5 µm/s or less. The times displayed are relative to the selected video frames. Scale bars are 20 µm. The feeding arteriole has a diameter of 21 µm, while the capillaries have an average diameter of 6 ± 2 µm. The complete video sequences of neutrophil migration within pulmonary capillaries and the tracking video displaying neutrophils with attached dragon tails are included in Movies S2 and S3.
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Figure 5. Discoid RBCs rapidly transit through the pulmonary microcirculation in a live C57BL/6 mouse. (A) Green channel. RBCs (green) were stained by iv administration of FITC Ter-119 mAb. (B) Red channel. Texas Red-conjugated dextran (red) was administered iv to visualize pulmonary capillaries and a feeding arteriole. The open arrow shows the direction of blood flow within the feeding arteriole. Note that Texas Red dextran fluorescence in the feeding arteriole decreases as a bolus of RBCs transits through it. (C) Merged dual color image. A t = 0 s, RBCs (green) can be seen traveling rapidly through the upper portion of the feeding arteriole (RBC transit is so rapid that their shape is distorted and they appear as green streaks in the image). Over the next 2 s, a bolus of RBCs (green) travels down the feeding arteriole and rapidly disperses into the capillaries. Since RBC transit is temporarily slowed at the arteriolar junction, the discoid shape of RBCs can be seen within the branches of the arterioles and within the capillaries. Dotted circles demonstrate that the presence of RBCs within capillaries is associated with dark spots or exclusion of the vascular dye within the capillaries. Alveoli are marked by asterisks. The times displayed are relative to the selected video frames. Scale bars are 20 µm. The feeding arteriole has a diameter of 29 µm, while the capillaries have an average diameter of 6 ± 2 µm. The complete video sequence of RBC trafficking within the pulmonary microcirculation is included inMovie S4.
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Figure 6. Post-acquisition spectral unmixing improves contrast of tricolor TPE images. Tricolor images of RBCs (green) and neutrophils (red) in the pulmonary microcirculation (purple) of a live C57BL/6 mouse (A) before spectral unmixing and (B) after spectral unmixing performed with NIS Elements software. The green channel shows RBCs stained with iv administration of FITC Ter-119 mAb. Spectral unmixing has served to reduce some tissue autofluorescence in the green channel, visible as a green haze in the original image A. The red channel shows neutrophils stained with iv administration of Alexa Fluor 546 Gr-1 mAb. The red channel also includes substantial bleed through from the intravascular fluorochrome (far red channel; purple) in the original image A. Spectral unmixing partially eliminates the bleed through, enabling visualization of neutrophils (red). After spectral unmixing, neutrophils appear as dark objects on a purple background far red (purple) channel. Three characteristic neutrophils are marked by the dotted circles. The merged tricolor images show that spectral unmixing leads to an improvement in visualization of both intravascular RBCs and neutrophils. Alveoli are marked by asterisks. Scale bars are 20 µm.
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Figure 7. RBC and neutrophil trafficking in the pulmonary microcirculation of live BERK non-sickle control and BERK SCD mice is similar in the absence of an inflammatory stimulus. RBCs are shown in green, neutrophils in red, and the pulmonary microcirculation in purple. Alveoli are marked by asterisks and open arrows denote the direction of blood flow through feeding arterioles. (A) Cellular trafficking in the lungs of a live BERK non-sickle control mouse. Blood flow through the pulmonary arteriole is rapid, as seen by the green streaks of RBCs. Some neutrophils are seen slowly transiting, while others rapidly transit through capillaries and arterioles. Dotted circles highlight a hopping neutrophil rapidly transiting within a capillary from t = 0 s to t = 0.5 s. Dashed circles highlight another neutrophil that quickly exits the feeding arteriole and enters a capillary from t = 18 s to t = 18.5 s. The feeding arteriole has a diameter of 41 µm, while the capillaries have an average diameter of 5 ± 2 µm. (B) Cellular trafficking in the lungs of a live BERK SCD mouse. Perfusion in the SCD mouse is similar to the control non-sickle mouse shown in A. RBCs are visible as green streaks in the feeding arteriole. Dotted circles mark a hopping neutrophil as it transits quickly through capillaries over a period of 1.5 s. Dashed circles mark another hopping neutrophil as it exits the feeding arteriole and enters the capillaries surrounding an alveolus. Slowly transiting neutrophils were also observed in the pulmonary capillaries of the SCD mouse. The feeding arteriole has a diameter of 41 µm, while the capillaries have an average diameter of 7 ± 2 µm. The times displayed are relative to the selected video frames. Scale bars are 20 µm. The complete video sequence of RBC and neutrophil trafficking within the pulmonary microcirculation of a live BERK non-sickle control and live BERK SCD mouse are included in Movies S5 and S6.

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