Name: in vivo two-photon imaging of megakaryocytes and proplatelets in the mouse skull bone marrow
Uploaded: 2021-07-28T14:30:00
Description: Watch this Scientific Journal Video about In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow at JoVE.com

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 D&#233;veloppement en M&#233;decine et Sant&#233; Publique) for its support in the acquisition of the two-photon microscope. AB was supported by post-doctoral fellowships from Etablissement Fran&#231;ais du Sang (APR2016) and from Agence Nationale de la Recherche (ANR-18-CE14-0037-01).

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 (Comit&#233; R&#233;gional d&#39;Ethique en Mati&#232;re d&#39;Exp&#233;rimentation Animale Strasbourg, Animal Facility agreement N&#176;: G67-482-10, project agreement N&#176;: 2018061211274514).
1. Preparation of mice and insertion of a catheter in the jugular vein
NOTE: Here, male or fem...

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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...

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 adult mice has a thickness of a few tens of microns and is sufficiently transparent to allow visualization of fluorescent cells in the underlying bone marrow2.
Ensuing studies applied this procedure to evaluate proplatelet formation under various conditions and to decipher the underlying mechanisms3,4,5,6. These studies provided definitive evidence that megakaryocytes dynamically extend protrusions, called proplatelets, through the endothelial barrier of the sinusoid vessels (Figure 1). These proplatelets are then released as long fragments that represent several hundred platelets in volume. The platelets will be formed after the remodeling of the proplatelets in the microcirculation of downstream organs, notably in the lungs7. To date, however, the precise process and molecular mechanisms remain subject to debate. For instance, the proposed role of the cytoskeletal proteins in the elongation of proplatelets differs between in vitro and in vivo conditions3, and differences in proplatelet formation have been demonstrated under inflammatory conditions6. Complicating things further, a recent study disputed the proplatelet-driven concept and proposed that in vivo, platelets are essentially formed through a membrane-budding mechanism at the megakaryocyte level8.
This paper presents a protocol for the observation of megakaryocytes and proplatelets in the bone marrow from the skull bone in living mice, using a minimally invasive procedure. Similar approaches have been previously described to visualize other marrow cells, notably hematopoietic stem and progenitor cells9. The focus here is on the observation of megakaryocytes and platelets to detail some parameters that can be measured, notably proplatelet morphologies and platelet velocity. This protocol presents how to insert a catheter into the jugular vein to inject fluorescent tracers and drugs and observe through the skull bone. The calvarial bone is exposed using minor surgery so that a ring is glued to the bone. This ring serves to immobilize the head and prevent movements due to breathing and form a cup filled with saline as the immersion medium of the lens. This technique is well suited to i) observe in real time the sinusoid geometry and megakaryocytes interacting with the vessel wall; ii) follow megakaryocytes in the process of proplatelet formation, elongation, and release; and iii) measure platelet movements to monitor the complex sinusoid blood flow. Data obtained using this protocol have been recently published3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GNU Octave software</td>
			<td>GNU Project</td>
			<td></td>
			<td>https://www.gnu.org/software/octave/</td>
		</tr>
		<tr>
			<td>&#160;Histoacryl 5 x 0, 5 mL</td>
			<td>Braun</td>
			<td>1050052</td>
			<td>injectable solution of surgical glue</td>
		</tr>
		<tr>
			<td>HyD hybrid detectors Leica Microsystems 4tunes</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>GNU project</td>
			<td></td>
			<td>Minimum version required</td>
		</tr>
		<tr>
			<td>Imalgene/Ketamine 1000 fl/10 mL</td>
			<td>Boehring</td>
			<td>03661103003199</td>
			<td>eye protection</td>
		</tr>
		<tr>
			<td>Leica SP8 MP DIVE microscope equipped with a 25x water objective, numerical aperture of 0.95</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>simultaneous excitation of AlexaFluor-488 and Qtracker-655</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>https://www.mathworks.com/</td>
		</tr>
		<tr>
			<td>Ocrygel 10 g</td>
			<td>Laboratoires T.V.M.</td>
			<td>03700454505621</td>
			<td>Silicon dental paste blue and yellow</td>
		</tr>
		<tr>
			<td>Picodent twinsin speed</td>
			<td>Rotec</td>
			<td>13001002</td>
			<td></td>
		</tr>
		<tr>
			<td>Qtracker 655 vascular label</td>
			<td>Invitrogen</td>
			<td>Q21021MP</td>
			<td>injectable solution</td>
		</tr>
		<tr>
			<td>Resonant scanner, 8 or 12 kHz</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun Xylazine 2% fl/25 mL</td>
			<td>Bayer</td>
			<td>04007221032311</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue gel</td>
			<td></td>
			<td></td>
			<td>to glue the ring to the bone</td>
		</tr>
		<tr>
			<td>Surflo IV catheter - Blue 22 G</td>
			<td>Terumo</td>
			<td>SR-OX2225C1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Saph pulsing laser (Coherent) (femtosecond)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon imaging of megakaryocytes and proplatelets in the mouse skull bone marrow

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.

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...

Watch this Scientific Journal Video about In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow at JoVE.com

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

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

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

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

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