Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

This research focuses on understanding vascular dynamics in the bone marrow macroenvironment using intravital microscopy, aiming to reveal how blood vessels respond to normal and pathological hematopoiesis. We use advanced imaging technologies like the photomicroscopy, transgenic animal models, and analyze a tracer to visualize and quantify dynamic vascular changes in living tissue over time. The key advancement is a refinement of our protocol to improve animal welfare and align with the three Rs.Our longitudinal approach minimizes the surgical steps and significantly reduce the number of animals needed for our study.

Our protocol addresses the need for nontoxic, long-term intravital imaging by using a two-photon microscope, which allows deep tissue penetration and high resolution visualization of cell behavior and interaction. Using the 3D printed head implant will make it very easy to find the area of interest during longitudinal imaging session without aiming to change the height, or angle, or the order. We are currently using this protocol to trace specific endothelial subtypes and their response to leukemia development and drug treatment to better understand their contribution on disease pathogenesis.

To begin, launch the segmentation software and import the micro-CT DICOM files to isolate the skull. Perform threshold-based segmentation to separate the bone tissue from surrounding structures. Manually adjust complex or noisy regions to refine the segmentation.

In 3D Slicer, import the DICOM files and load the dataset. Observe the CT slices in the axial, sagittal, and coronal slice views. Open the segment editor and click Add to create a new segmentation, then click add again to create a new segment for thresholding.

In the segment editor panel, click on the threshold effect. Adjust the lower and upper threshold sliders or enter numeric values to highlight the segmented region in red. To export the segmented skull as an stl file, switch to the segmentation module.

Ensure the thresholded segment is selected. In the export/import models and labelmaps section, set export type to models and file format to stl, then click export to save the file. For preparation of the skull model in CAD, first import the simplified skull model in stl format into the CAD software as a new part or model.

In the median plane, create an axis tangential to the calvaria, then save the prepared skull model. Create a sketch in the calvarial plane and design a pear shaped spline from AP plus 6.5 to minus two. With a width of six millimeters at AP 0.0, this will be the observable surface contour.

Design the tail of the implant ensuring it conforms to the skull structure for proper fixation while respecting the available volume. Finally, create a protective cover to safeguard the observation window when not in use. Place an anesthetized mouse on a heating pad set to 37 degrees Celsius and visually monitor the respiratory rate.

Shave the mouse's head with an electric razor. Then apply a drop of ophthalmic gel to the eyes of the mouse. Ensure all hair is removed to prevent imaging artifacts and reduce the risk of wound infection.

Now, use a pair of sterile forceps and scissors to make a small incision in the central portion of the scalp to expose the central bone scar. Remove the connective tissue between the skull and the scalp. Mix sufficient dental cement in a Petri dish to make a paste.

Quickly apply it to the bottom of the head fixation implant. Without allowing dental cement to enter the imaging area, place the head holder onto the exposed skull and let it set. Switch on the Isoflurane flow toward the stereotaxic mask of the microscope.

Now, bring the mouse rapidly to the microscope. Carefully insert the mouse's teeth into the mask to ensure proper isoflurane penetration by lifting its nose with one hand. While holding the mouse with one hand, gently slip the dovetail of the head fixation implant into the fixation holder using the other hand.

Secure it with a half turn of the screw knob. Next, introduce a rectal probe pre-embedded with a water-based gel to monitor the mouse's temperature. Fill the head imaging chamber with a large amount of water-based gel, or PBS.

Lower the water immersion objective until it is fully immersed for optimal excitation and signal detection during two-photon excitation. Move the x, y stage and the z drive to focus on the central bone in the scaffold scar. Identify the tissue marked by the bone surface and central vein.

Switch off the light in the room and close the box around the microscope stand. After activating the non-TOSCAN detectors. Set PMT1 to detect second SHG at 423 to 461 nanometers, high D2 for GFP at 485 to 548 nanometers, and high D3 for TD D'Amato at 551 to 645 nanometers.

Leave the offset at zero. Now, set the PMT SHG gain to 850 volts and the high D gains at 100%Increase the infrared laser power until an image with a dynamic range of 200 gray levels is visible on the lowest of the three channels. In the acquisition software, locator region containing bone marrow pockets, indicated encased within the bone surfaces.

To identify different regions of interest, or ROIs, activate Loss Navigator and create an overview using the spiral mode. Click on the single image icon to record selected ROI positions and rename each position in the task list. To acquire a Z stack volume, select Z stack mode and define the step size at three micrometers.

Deselect the same stack size for all regions option, then press begin to start and end to finish. After capturing, click redefine stack. Press start to start image collection and save the images in the appropriate folder.

To measure a dynamic feature, such as vascular permeability, change the acquisition mode to XYZT to acquire a time lapse. In the T module, adjust the time interval to three minutes and the duration to one hour. Now, inject 100 microliters of 70 kilodalton Dextran TRITC intravenously at a concentration of four milligrams per milliliter, and start the acquisition.

After transferring the mouse to a surgery mark on a 37 degrees Celsius heating pad, apply intrasite gel to the skull to keep it hydrated. Close the imaging area of the head implant with the specific cover and carefully secure the cover with a screw. Place the mouse into the heating box set to 37 degrees Celsius and wait for it to awaken.

Once the mouse has fully recovered, transport to the animal facility and house it in a clean cage with hydrogel and environmental enrichment. For longitudinal acquisitions, place a small drop of ophthalmic gel on the eyes of a mouse with a head holder already installed. Obtain the overview image as demonstrated earlier.

If necessary, realign the previous and new images using the load and align image module. Open the screen capture image to mark positions in their original place. The CAD model of a titanium head fixation implant was designed to conform to the anatomical structure of the skull, ensuring stable attachment to the microscope stage for cellular level imaging.

The implant was successfully attached to the mouse skull and enabled stable imaging over time, while allowing the mouse to maintain normal activities. The calvarial bone marrow vasculature was visualized using a tile scan, revealing a complex network of arterioles, transition capillaries, and sinusoids. Quantitative vessel analysis demonstrated segmentation of vascular structures, allowing measurement of vessel diameter, length, and straightness, and their correlation.

Longitudinal imaging captured progressive remodeling of calvarial vasculature during leukemia progression with increased vascular density observed over time. Vascular permeability was assessed dynamically, showing differential retention of fluorescent dye over time, displaying differential permeability of different bone marrow vessels.