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

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.