Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

The overall goal of this procedure is to create a consistent and reproducible human brain tumor model that can be used for the accurate testing and comparison of novel therapeutic strategies. This is accomplished by preparing the animal for surgery and locating the correct site for injection. Once located, a hole is drilled in the skull and a syringe is lowered at the proper depth.

To obtain tumor growth, cells are then injected in a slow controlled manner, followed by a delayed and gradual withdrawal of the needle. Ultimately, a consistent mouse model with tumor growth histopathologically similar to the human condition can be developed and growth rates can be measured by Lucci based in vivo imaging. The precision of this technique is vital to understanding new therapeutic strategies for human brain tumors.

Variable localization of tumor injection can lead to changes in tumor growth that are due to microenvironment, drug delivery, or other things that vary greatly amongst different areas of the brain. Visual demonstration of this method is important because the steps involving mouse positioning and injection site localization require three dimensional manipulations, which are not easily described in written or graphical format. To begin clean and decontaminate a mouse stereotaxic frame and all other equipment needed for surgery.

Using settings optimized for the model chosen program, the injection parameters, and other variables for the operation of the syringe pump. Next, arrange autoclave surgical tools in the work area include at least two 1.0 millimeter dental drill bits for serial procedures. Disinfect surgical instruments with 70%ethanol, followed by 30 seconds in a bead sterilizer between each animal or as advised by your IACUC protocols.

Lastly, gather together all of the medical supplies and disposable materials required A fine tip, permanent marker for marking the skull should be decontaminated and dedicated for surgical use. After trypsin resus, suspend the cells in PBS and pellet by centrifugation Resus. Suspend the cells in approximately five milliliters of serum free dmem per 100 millimeter plate.

Next, count viable cells by using a hemo, cytometer, or an automated cell counter. After counting, adjust the live cell density by pelleting the cells and resus suspending an SF dmm. Keep the cells chilled on ice or a chill pack, but do not allow the cells to freeze.

Mix gently by finger flicking induce animal anesthetic and confirm that the animal is adequately sedated by checking for writing reflex or lack of activity. Once anesthetized, cover the mouse with gauze pads for warmth and monitor the animal regularly throughout the procedure. Next, position the mouse in a stereotaxic frame.

Using the pallet bar and ear bars, try to keep the ear to eye surface as horizontal as possible. Lastly, lubricate the eyes with ophthalmic ointment and clean the skin between the ears and eyes twice with alternating applications of a povidone iodine antiseptic, and 70%ethanol. After injecting an analgesic subcutaneously, confirm that the animal is fully anesthetized by checking the toe pinch reflex.

Immediately prior to skin incision, make a small incision to expose the BMA and the site of an injection. A diagonal cut from a point between the midline axis and the right eye towards the right ear allows axis to both the bgma and the injection site. Retract the skin and use a sterile cotton swab to dry the surface of the skull.

Blot the bone with another swab, moistened with hydrogen peroxide. To visualize bgma, lock an empty syringe with needle into the micro pump and maneuver the tip directly over the bgma. Zero the coordinates on the alignment console.

Move the needle to 2.5 millimeters lateral and 1.5 millimeters anterior with respect.Torema. Using the control knobs on the stereotaxic unit, raise the syringe slightly and mark the exact location on the skull with a dedicated felt tip.Pen. If the outer surface of the skull is no longer level with bgma in the dorsal plane, reset the dorsal ventral reading to zero to ensure that the depth of the injection is correct.

Drill a small hole into the skull with a handheld rotary drill outfitted with a sterile dental tip. Hold the drill at an angle to the skull and very gently touch the tip to the bone. Stop short of drilling completely through the bone.

The cleanest injection with the least trauma is obtained. When the skull is pierce with the syringe needle during cell injection, remove bone dust with a cotton swab moistened in PBS or saline. Return the syringe to the pump and lower the needle straight down to the bur hole.

To confirm location, gently mix the cell suspension and draw the cells into the syringe. Using the pump controller, avoid bubbles and clumps and wipe the needle with an alcohol swab. To remove contaminating cells on the exterior surface, lower the needle to the level of the skull surface.

Then slowly lower the needle. Pierce the full thickness of the skull and penetrate the brain to a depth of four millimeters ventral over the course of one minute, before withdrawing the needle slowly to 3.5 millimeters ventral, confirm that the correct parameters are entered into the pump controller and press run.Stop. To autoinject the cells, watch the syringe and ensure that the plunger is moving into the barrel.

Leave the needle in place for one to two minutes before very slowly withdrawing the needle from the tissue. The needle should be removed over the course of five minutes. Use a cotton swab to blot the area around the bur hole and leave the edges of the skin open to allow the bone to dry.

Remove the syringe from the pump and quickly rens it with sterile water and ethanol. Next, apply sterile bone wax to the bur hole and use the wooden end of a sterile cotton swab to tamp the wax onto and into the bone. If the surface is dried adequately, it should stick After suturing the wound, reduce the isof fluorine to 0%and remove the mouse from the stereotaxic unit.

Place the animal onto a heated surface and observe until fully recovered. It is important to clean all instruments and equipment using 70%ethanol and a bead sterilizer or disinfect between each animal. The injected mice should be monitored for two days after the procedure for signs of pain, infection, or other complications.

Evaluate the mice for clinical signs of disease paralysis, decreased activity, weight loss, seizures, or general illness at desired time. Points after injection, monitor the tumor development by either magnetic resonance imaging or in vivo imaging systems. These MRIT two weighted images compare a tumor derived from U 87 cells with the tumor derived from U2 51 cells.

Tumor volumes plotted over time shows a reproducible window of tumor development and growth that is consistent in all experiments. An IVIS image of U2 51 lucifers Transduced GBM cells injected intracranial into nude mice, reveals that the mouse on the left was successfully injected and shows a very strong vocalized signal in the desired location. While the mouse on the right demonstrates an unsuccessful result from improper injection location or technique.

Tumor size is estimated from lucifers activity in the brain region of interest and plotted over time. The blue line corresponds to the mouse with a successful intracranial injection while the red line corresponds to the mouse with tumor cell displacement to the spine after h and e staining the tumor from U 87 GBM cells shows an area of dense tumor growth and adjacent normal brain tissue with microscopic invasion of malignant cells. This section of the center of a tumor derived from U2 51 GBM cells very closely replicates the bizarre histopathology with multi-nucleated malignant cells seen in typical human GBM.

When attempting this procedure, it is important to remember that tumor model reproducibility depends on the precise and secure positioning of the mouse's head and consistent localization of the injection site. Also, a gradual rate and consistent pressure of injection is essential. This technique allows researchers in the field of neuro-oncology to explore new therapeutic strategies for human brain tumors while removing some of the variability that is compounded by imprecise localization of brain tumor cell injection.