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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(113), e542205 (2016).</li><li>Guesmi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-color+deep-tissue+three-photon+microscopy+with+a+multiband+infrared+laser.">Dual-color deep-tissue three-photon microscopy with a multiband infrared laser.</a> Light Science &amp; Applications. 7, 12 (2018).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-photon+imaging+of+mouse+brain+structure+and+function+through+the+intact+skull.">Three-photon imaging of mouse brain structure and function through the intact skull.</a> Nature Methods. 15 (10), 789-792 (2018).</li><li>Kim, J., Bixel, M. 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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 ...

This method allows the visualization in real time of different events, taking place in the bone marrow of the skull mouse, such as megakaryocytes, extending proplatelets inside the sinusoid vessels. The main advantage of this technique is a minor surgery needed minimizing the inflammatory reaction, allowing an in vivo visualization of events in the almost factory conditions. To begin, transport the mouse to the imaging room.Switch on the microscope and computer and set up the required parameters. Once the mouse has completely anesthetized place the mouse on a heated plate, warmed to 37 degrees Celsius for all following manipulations, shave the head and throat of the mouse and apply the gel to the eyes to prevent dryness. Place the mouse on its back and fix the anterior legs with surgical tape to stretch the throat.After disinfecting the throat, make a 0.7 to 1 centimeter incision over the right or left jugular vein using scissors and stretch the adjacent connective tissue to expose the external juggler vein on top of the pectoral muscle. Fill the catheter with warm, sterile physiological saline. Penetrate the catheter through the pectoral muscle and then insert the catheter into the vein.Gently remove the needle, connect the syringe with the fluorescent tracers and inject the dead volume of the catheter. Place a drop of surgical glue to stabilize the catheter. Carefully return the mouse to a prone position.Disinfect the scalp with 70%ethanol with a paper towel while removing the loose hair. Use sterile fine scissors and tweezers to make a T-shaped incision at the midline up to one centimeter in between the ears on the scalp to expose the calvarium. Expose the skull with tweezers, then use scissors and tweezers to carefully remove the periosteum.Use a sterile cotton swab soaked and physiological saline to remove all the periosteum and any debris or hair which could alter the imaging. Rinse the skull with saline to remove any blood traces and rapidly dry the bone with a cotton swab. Apply the glue gel to the ring, place the ring on the exposed bone and maintain it for a few seconds to allow the ring to firmly attach to the skull.Lightly moisten the skull with a saline wedded cotton swab. Prepare the Silicon dental paste by carefully mixing the blue and yellow components at a one-to-one ratio. Seal the ring by applying the dental paste to prevent leakage during imaging.Carefully remove any dental paste or glue that may have entered the ring. Then fill the ring with saline to check the leakage. Place the mouse on the support and put a folded compress under the animal's head to raise the head and prevent detachment of the ring from the skull when attached to the holder.Screw the ring on the block holder. Place the support and mouse assembly in the heated microscope chamber. Set the appropriate laser wavelengths for the chosen four fours and recovery of the emitted lights.Using the inter juggler catheter inject 50 microliters of 0.2 micromolar solution of the fluorescent tracer to label the vasculature. Place the microscope stage with the support and mouse under the objective of the microscope. Ensure that the ring is always filled with saline and refill it if needed, and immerse the objective and saline.Use epifluorescence to locate the bone marrow vessels and observe the megakaryocytes aligned along the sinusoid vessels. For long acquisitions, set up a 3D space-time acquisition with a 384 by 384 pixel image using an eight kilo Hertz resonance scanner, and bidirectional mode. Use line averaging with good resolution, then choose the optimized Z step size and set up the time interval needed before starting the acquisition.For short and rapid acquisitions, choose a space, time acquisition type, and minimize image size. If required, adjust the image to the vessel by rotating the imaging field. Use the highest available scanning speed and bidirectional scanning.Minimize the line averaging to find the optimal compromise between image definition and rapidity of the acquisition. Acquire only one Z plan and minimize the time interval for acquisition. For measuring the platelet velocity 10 to 20 seconds of acquisition should be sufficient.The fluorescent tracer was intravenously administered to image the anastomosed marrow sinusoid vessels in the skull bone marrow, with the flow direction depicted by the arrow. The fluorescent platelet velocity was recorded in each vessel branch, and variation was observed. The sinusoid vessels present complex flows due to the anastomoses, with the presence of flow reflow and even stasis.Arrows indicated the opposite direction of flow at the bifurcation. The platelet velocity was also measured for left and right vessel branches indicated irregularity over time in each vessel branch with phases of acceleration, stasis and deceleration. Different morphologies of proplatelets were observed, including proplatelets with irregular margins, Then en longed proplatelets and thick and short proplatelets.When megakaryocytes extended the proplatelets in the areas of complex flows, the proplatelets tossed from one vessel branch to the other, according to the flow direction. Upon stopping the blood flow, the proplatelets relaxed and the mouse unexpectedly underwent cardiac arrest, indicated the importance of hydrodynamic forces in proplatelet elongation. The width and length of the proplatelets were also measured mean width of 5.2 micrometers and a mean maximal length of approximately 185 micrometers were observed.Plotting the proplatelet length as a function of time allows the visualization of the behavior of proplatelets during phases of elongation, stasis, or even retraction. The mean elongation velocity of approximately 10 micrometers per minute was observed. Installation of the catheter, allowed the injection of drugs to study the effects on proplatelet formation or other events of interest in real time.

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3.9K vistas.  Université de Strasbourg. Este método permite la visualización en tiempo real de diferentes eventos, que tienen lugar en la médula ósea del cráneo del ratón, como los megacariocitos, extendiendo los proplatatos dentro de los vasos sinusoides. La principal ventaja de esta técnica es una cirugía menor necesaria minimizando la reacción inflamatoria, permitiendo una visualización in vivo de los eventos en las condiciones casi de fábrica. Para comenzar, transporte el ratón a la sala de imágenes.Encienda...