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In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Published: July 28th, 2021



1INSERM, EFS Grand Est, Université de Strasbourg

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.

Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

Platelets are produced by megakaryocytes-specialized cells located in the bone marrow. The precise way megakaryocytes release platelets in the circulation has long remained unclear owing to the technical challenge in observing real-time events through the bone. Two-photon microscopy has helped overcome this challenge and led to major advances in understanding the platelet formation process. The first in vivo megakaryocyte observations were made by von Andrian and colleagues in 2007, with the visualization of fluorescent megakaryocytes through the skull1. This was possible because the bone layer in the frontoparietal skull of young adul....

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All animal experiments were performed in accordance with European standards 2010/63/EU and the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg, Animal Facility agreement N°: G67-482-10, project agreement N°: 2018061211274514).

1. Preparation of mice and insertion of a catheter in the jugular vein

NOTE: Here, male or fem.......

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Using this protocol, the fluorescent tracer, Qtracker-655, was intravenously administered to image anastomosed marrow sinusoid vessels in the skull bone marrow and the flow direction as depicted by the arrows (Figure 4A, left). Using mTmG mice, eGFP-fluorescent platelets were recorded over 20 s in each vessel branch, and their velocity was measured using ImageJ and GNU Octave software (Figure 4A, right). Note the heterogeneity in flow velocity and direction. Sin.......

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The mechanisms of platelet formation are highly dependent on the bone marrow environment. Hence, intravital microscopy has become an important tool in the field to visualize the process in real-time. Mice with fluorescent megakaryocytes can be obtained by crossing mice expressing the Cre recombinase in megakaryocytes with any floxed reporter mice containing a conditional fluorescent gene expression cassette. Here, mTmG reporter mice were used (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo10) cro.......

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The authors would like to thank Florian Gaertner (Institute of Science and Technology Austria, Klosterneuburg, Austria) for his expert advice on two-photon microscopy experiments at the time when we established the technique in the lab, and Yves Lutz at the Imaging Center IGBMC /CBI (Illkirch, France) for his expertise and help with the two-photon microscope. We also thank Jean-Yves Rinkel for his technical help and Ines Guinard for the drawing of the schema in Figure 1. We thank ARMESA (Association de Recherche et Développement en Médecine et Santé Publique) for its support in the acquisition of the two-photon microscope. AB was supported by post-docto....

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Name Company Catalog Number Comments
GNU Octave software GNU Project
 Histoacryl 5 x 0, 5 mL Braun 1050052 injectable solution of surgical glue
HyD hybrid detectors Leica Microsystems 4tunes Leica Microsystems
ImageJ GNU project Minimum version required
Imalgene/Ketamine 1000 fl/10 mL Boehring 03661103003199 eye protection
Leica SP8 MP DIVE microscope equipped with a 25x water objective, numerical aperture of 0.95 Leica Microsystems simultaneous excitation of AlexaFluor-488 and Qtracker-655
Matlab MathWorks
Ocrygel 10 g Laboratoires T.V.M. 03700454505621 Silicon dental paste blue and yellow
Picodent twinsin speed Rotec 13001002
Qtracker 655 vascular label Invitrogen Q21021MP injectable solution
Resonant scanner, 8 or 12 kHz
Rompun Xylazine 2% fl/25 mL Bayer 04007221032311
Superglue gel to glue the ring to the bone
Surflo IV catheter - Blue 22 G Terumo SR-OX2225C1
Ti:Saph pulsing laser (Coherent) (femtosecond) Coherent

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