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The overall goal of this procedure is to obtain high resolution single cell images of bone marrow derived progenitor cells following their recruitment patterns to normal brain and brain tumor associated vasculature with time. This is accomplished by removing the tibia and femur from donor mice to obtain fluorescent bone marrow progenitor cells, and then injecting those cells into a radiated recipient mice. The second step is to generate intracranial GB M xenografts in the chimeric mice inside a cranial window chamber.

Next, the mice undergo a variety of treatment regimes including stereotactic guided radiation. The final step is to image the mice on a two photon confocal microscope. Ultimately, this methodology can be used to dynamically study the mechanisms of vascularization intracranial in a variety of models.

The main advantage of this technique over existing methods like histopathology of ex vivo tissue, is that we can study critical dynamic information related to vessel formation in the brain. As such, we can answer key questions surrounding the mechanisms of intercranial neovascularization. Though this method can be used to provide insights into intracranial neovascularization, it can also be applied to other systems and disease processes in the brain, such as intracranial ischemia or stroke neurodegenerative disease and mechanisms of tumor cell metastases to the brain.

For the purpose of this video, we were unable to follow strict aseptic technique. As such, biosafety hood is an excess of 12 inch opening and the animals are not draped, allowing the viewer a clearer image and better access to orientate the procedures for experimental work. Aseptic technique should be strictly followed.

Begin this procedure by confirming the fluorescence expression of the GFP donor mouse and euthanize it according to the institutional animal care committee guidelines. Next, clean both hind limbs and remove the tibia and femur while working in aseptic conditions. Strip all excess tissue from the bones.

Then remove the end plates from both ends of the four extracted bones. Flush them with a 22 gauge needle and one milliliter of sterile PBS. They will turn clear once all of the bone marrow has been flushed out.

Afterward, mix the extracted bone marrow suspension. Well, it should contain approximately 20 million cells, which is enough for three host mice reconstitutions. Next, prepare 3 27 gauge tuberculin needles and draw 300 microliters of the bone marrow suspension into each of them.

Whilst the mouse is positioned in the restrainer. Mark the dorsal tail surface to orient yourself. Then inject the suspension into the lateral tail vein of three previously irradiated.

Nod skid mice in this procedure working under aseptic conditions. Anesthetize the chimeric bone marrow nod skid mice, and administer iacuc approved preemptive analgesia. Apply tear gel to prevent corneal dehydration.

Clean the head first with Betadine solution, then with alcohol and remove the excess hair. Then clean the underlying scalp further with alcohol. Make an incision from the midpoint of the ears to just above the eyes.

Remove the scalp at either side of the first incision to expose about five millimeters of the underlying skull. Then identify the anatomical landmarks of the skull surface. Next, identify and lift the periosteum by injecting 2%lidocaine with epinephrine solution.

Subsequently dissect the periosteum away from the skull surface with a sharp instrument. Then use a 2.7 millimeter TR refine drill to weaken a 2.7 millimeter circular bone flap on the right hand hemisphere of the skull equidistant from both Lambda and bgma. Do not penetrate the bone with the drill in order to protect the underlying dura and tissue.

Now, remove the weakened bone flap using the dissection, tweezers, and dental hook with deliberate but controlled force. Next, load a 30 gauge Hamilton syringe with 10 microliters of cell suspension and place the needle onto the stereotactic frame. Then place the mouse on the stereotactic frame.

Align the needle to the center point of the window generated after that, lower the needle till it touches the cortical surface. Then reset the digital coordinates. Now lower the needle 3.2 millimeters into the cortical tissue.

Then retract 0.2 millimeters and inject at three millimeters. Depth injection should be carried out at a rate of 10 microliters per minute. When the injection is completed, leave the needle in place for one minute before retracting it slowly.

Then remove the mouse from the frame. Subsequently, keep the brain surface hydrated. Using drops of sterile PBS.

Place a three millimeter cover slip on the brain surface to seal the 2.7 millimeter window. Then dry the surrounding skull bone. Next, apply vet bond to glue the scalp tissue to the skull bone and keep the cover slip in place.

Do not apply too much as it will leak under the glass and decrease the imaging potential. Next, mix the fresh dental acrylic powder and solution in the hood and apply the mixture over the vet bond using a 22 gauge needle. To ensure a tight seal, apply the acrylic to slightly overlap with the edge of the glass cover slip, but not to cover the glass cover slip.

In this step, place an anesthetized mouse into a custom head restrainer inside the house designed XAD 2 25 stereotactic irradiator. Obtain a 360 degree cone beam CT scan with the x-ray tube running at 40 peak kilo voltage and 0.05 milliamps through a two millimeter aluminum filter. Use the image to position the stage such that the radiation ISO center is aimed to the right hemisphere and is central in the dorsal ventral direction.

There are a wide variety of collimators available for targeting the radiation, which demonstrates the adaptability of the model. In this case, we use eight millimeter by 11 millimeter for hemispheric irradiation. Insert the hemispheric collimator into the X RAD 2 25 stereotactic irradiator.

Obtain the single CT orthogonal images from both the top and the bottom through the collimator to ensure correct positioning of the head by locating the anatomical bone structures and manually fine tuning the stage position on the monitor. Change the XAD 2 25 IRRADIATOR aluminum filter to a 0.93 millimeter copper treatment filter. Then run the x-ray tube program at 225 peak illa voltage and 13 milliamps and administer half of the radiation dose from the top in an AP direction.

Return the gantry to the bottom position and administer the second half of the radiation dose from the bottom in a PA direction. In this procedure, anesthetize the previously generated intracranial window mouse and clean the window using alcohol spray. Set up the channels on the confocal microscope as determined by the fluorochromes integrated into the chimeric mouse generated.

Alternatively, reuse previous settings to reduce variability between imaging sessions. Inject any tags required for imaging to the lateral tail vein in this case, dextran for vasculature. Invert the mouse onto the custom-built head restrainer, supporting with moldable plasticine, ensuring the head is perpendicular to the laser and flat load the restrainer onto the movable microscope stage.

Turn on the first channel laser and position it to the center of the intracranial window by directly looking at the laser beam intersection point on the window of the chamber. Take an image of each channel for the entire window with a five x lens and use it as a map for other higher resolution images with 10 x and 20 x long range lenses. This schematic highlights the technical errors associated with the surgical process and demonstrates the effect each will have on the images produced.

Trapped air under the window will be visible as bubbles on the images. Acrylic buildup on the glass is autofluorescent in the far red channel and will block out part of the field of view. Similarly, dirt on the window during imaging shows up as autofluorescent flex in the images.

Even the perfect intracranial window can encounter problems once it is under the microscope. Breathing artifacts result in lined images whilst poor positioning of the mouse on the frame results in a segmented image. As the laser only crosses part of the tissue with post image processing, it is possible to add an additional channel for CFP tagged cells whereby GFP images can be subtracted from CFP images to reveal the true CFP signal.

In addition, utilizing the second harmonic generation autofluorescence characteristic of collagen four fibers, we are able to image the basement membrane of the vasculature. The availability of five fluorescent channels allows the user a large amount of flexibility and adaptability of the novel mirroring model described in this paper. While attempting this procedure, it is important to keep in mind the hazardous products used during the surgical procedures.

It is also essential to be meticulous and pay specific attention to detail to ensure that the brain tissue remains undamaged throughout the long-term imaging. After watching this video, you should have a good understanding of how to create a chimeric mouse with fluorescent progenitor cells, and in turn be able to capture high resolution realtime images of the intracranial vasculature following tumor cell implantation with or without therapeutic intervention.