An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

The overall goal of this procedure is to generate an orthotopic glioblastoma model suitable for intravital optical imaging in the mouse cerebral cortex. This is accomplished by first creating a cranial window above the parietal cortex. The next step of the procedure is to implant a glioblastoma S spheroid tumor into the superficial layers of the brain.

Parenchyma then sealing the dator and clogging the injection. Track forces intraparenchymal tumor development and maintains biophysical constraints. The final step is to seal a glass window in close contact with the brain surface to prevent scar tissue, thereby ensuring optimal clarity during imaging.

Ultimately, the results can show glioblastoma tumor progression, vascular remodeling, and the cellular microenvironment at single cell resolution through intravital two photon microscopy. We first had the idea of this method when we try to graft self suspension of self steroid without physical barrier to avoid cellular escape, and this resulted mainly in unphysiologic, in vitro like extra growth. This method can help key questions in the neuro oncology field, such as the role of vascular remodeling and the regulatory effects of cellular interactions during blato.

Much graph To implant the steroid begin by lightly sedating the mouse in a gas chamber, then fully anesthetize the mouse with an intraperitoneal injection of ketamine with xylazine. Perform the surgery in a warm room at 26 degrees Celsius and keep the mouse warm with a controllable heating pad. After applying eye ointments and shaving the scalp, place the mouse in a stereotactic frame with ear bars and a mouthpiece with the animal in position.

Clean the skin with 3%soap and 10%iodine solution. Now make a one centimeter long incision longitudinally in the middle of the scalp using scissors. Cut and remove the skin above the parietal bones with a scalpel blade, gently remove the periosteum above the skull.

Generously apply cyanoacrylates on top of the bone to generate a rough surface for later cement adherence. Now under a surgical microscope using a scalpel blade plan to drill the parietal bone at least a millimeter from the skull sutures to avoid hemorrhages and to finally remove the parietal bone over one cerebral hemisphere while drilling, add plenty of ice cold PBS with penicillin and streptomycin to prevent overheating. Bone fragments can be removed with forceps and wet gauze every minute or so, and then all can be removed when the drilling is finished.

Next, then the bone at the border of the craniotomy where the glass window will be sealed. Drill until bone piece gets weakly attached, and finally, slowly chisel the bone with forceps. Ultimately, the window should be against as much brain tissue as possible.

After the window frame is completed, test it with a dry alcohol cleaned five millimeter round glass cover slip. Continue chiseling if needed until the brain is squeezed flat with gentle pressure to the cover slip. Then clean the cover slip again and set it aside to open the dura mater.

Make a hole with a 26 gauge needle at the center of the craniotomy, avoiding Maine blood vessels. Be sure not to damage the brain parenchyma. Gently clean the dira mater with PBS and then cover it with moist tissue paper while preparing the s spheroid.

This procedure requires that the S spheroid injection system and a Petri dish be prepared in advance. First aspirate a round steroid that fits the capillary inner diameter from the Petri dish. The steroid should come along with roughly five microliters of culture medium, and it should fall down to the tip of the capillary under its own weight.

Remove the wet tissue paper used to cover the craniotomy and position the animal under the injection system. Lower the injection pipette until it touches the hole made in the dura mater. Then lower it again by 250 microns and wait 30 seconds.

Slowly inject the steroid using the piston of the microliter syringe, two to three microliters while sopping up the excess liquid with a thin piece of tissue paper. Wait 30 seconds and then gently lift the injection pipette 50 microns. Then wait another 30 seconds before lifting another 50 microns.

Keep repeating this pattern to bring the pipette to the surface. Now, confirm the presence of the steroid in the brain using a fluorescence microscope in a Petri dish. Prepare a viscous soaked bead solution of one to 300 micron diameter.

Cross links Dexter and gel beads and PBS. Wait a minute and transfer some of the hydrated beads to the bone adjacent to the craniotomy under microscopic control, choose one bead with a diameter similar to the size of the diameter opening. Cut the cross links DExT strand gel bead in two halves.

Using forceps, gently put a cross-linked dextra and gel hemi bead into the injection hole with a convex face towards the S spheroid. Staying aware of the implant's fragility, press gently on the bead until its flat surface aligns with the dura mater. Next, put a drop of cyanoacrylate glue on a glass slide and using a toothpick glue, a seal along the edges of the DExT strand bead.

Stamp the glue around quickly and accurately, or the bead may get glued to the toothpick. Do not use too much glue, but enough to induce a change in porosity of of the bead. As illustrated here on sample beads, This very delicate step consists entirely.

Seal the bead to the ter and surrounding parenchyma with just enough sac to not induce a bump of glute that would otherwise impede glass window placement and long-term optical clarity. Wait two minutes for the glue to dry and then clean the craniotomy in the surrounding bone with normal PBS. Now position the prepared cover slip above the craniotomy.

The edges of the cover slip must rest on the thin skull perimeter and the underlying dura mater must contact the glass or scar tissue can develop and impede optical clarity with the tissue. Absorb the PBS along the cover slip edges so that the solution does not cover the craniotomy. When applying a small amount of pressure in the center of the cover slip, this will ensure that bone, glass and brain are glued together at the region of interest.

Maintain light pressure at the center of the cover slip with forceps while gently applying cyanoacrylate at the border between the bone and the cover slip, the glue will spontaneously spread until it is repelled by the PBS solution. Within a minute, an effective seal is formed, but take extreme care not to move the cover slip until it is fixed or the surgery will fail due to glue leakage. If glue spreads on top of the glass, remove it with a micro scalpel blade using gentle pressure and spiral motions to maintain optical clarity.

Now, consolidate the glass fixation by applying dental cements on the edges of the cover. Slip toward the adjacent skull. Cover the whole exposed skull to the scalp using extra cement.

Build up sidewalls around the cover slip so that a pool of solution can be made over the glass. For the immersion objective, be aware that the dental cements will cure in less than 10 minutes. After removing the mouse from the stereotactic frame, lay it down in a cage with a warm tissue nest and proceed with a postoperative care regimen.

Tumor growth below the dura mater can be viewed for weeks after the implantation. This tumor's development was estimated over time using a fluorescence microscope, combining oblique illumination for brightfield reflectance imaging and epi fluorescence emission thresholding of the fluorescence easily gives access to the tumor area while superficial vasculature detaches as dark structures out of the white background of the visible light image. Alternately intravital two photon microscopy allows Z sectioning and orthogonal reconstructions of large fields of view with subcellular resolution such as dendrites.

The green shows cyan fluorescent protein expression, and neurons following tumor growth with two photon microscopy from day 16 to day 27. Some blood vessels highlighted by arrows were stable over the 11 days while the tumor margin outlined shifted 520 microns and the tumor's vasculature changed at the tumor margins. Detaching glioma cells could be seen from the tumor core as expected from an invasive tumor.

These cells emit cytoplasmic protrusions and use blood vessels as a matrix for their progression pointed out by the arrowheads paired with a fluorescent reporter. This method makes it possible to view such physical interactions between tumor cells and their environment as key regulators of disease progression. Unmastered, this method can be done in 45 minutes to one hour if it is performed properly Following this procedure.

Other preclinical imaging methods like fluorescence tomography X-ray, CT scanner MRI can be performed in order to answer additional questions like the infiltration of neoblastoma cell into the old brain.