Surgical Cisterna Magna Injection: A Method for Administering Tumor Cells Directly into Central Nervous System of Murine Model

The murine brain is lined by three meningeal layers - the outermost dural membrane, the middle arachnoid membrane, and the innermost pia membrane.

In the posterior region of the brain, the arachnoid and pia membrane are intervened by a cerebrospinal or CSF-filled subarachnoid space, called the cisterna magna.

To perform intracisternal injection of the tumor cells, begin by taking an anesthetized mouse model. Prep the mouse by removing the hair from the ventral surface of the head.

Now, place the mouse in the prone position and attach its head to a stereotaxic frame. Position the nose of the mouse, pointing it slightly downwards to level the spine with the cisterna magna.

Using scissors, make a small midline incision on the posterior end of the brain, at the ears level.

Carefully pull aside the superficial muscle layer. The cisterna magna can be visualized as an inverted triangle covered by the transparent dural membrane.

Next, take a syringe containing circulating tumor cell suspension.

Keeping the surrounding musculature retracted, insert the syringe under the dura membrane, piercing through the arachnoid membrane. This step allows the injection of tumor cells directly into the CSF present in the cisterna magna.

Finally, close the incision site by applying wound clips, and allow the mouse to recover.

Begin by injecting the mouse subcutaneously with 1 milligram per kilogram sustained-release buprenorphine. After anesthetizing the mouse, prepare it for surgery. Clip the surgical site with enough border area to keep fur from contaminating the incision site.

Shave the fur of the entire surface of the head and prepare the skin using sterile technique. Then, saturate the site with germicidal skin antiseptic, working from the center of the site to the periphery, and allow it to dry.

Position the body to ensure the spine is kept level with the cisterna magna. While applying slight traction to the tail, place tape at the tail base to secure it.

Apply either a sterile drape or a sterile adhesive-backed plastic drape material to protect the surgical site from contamination. With the neck in full extension, run the surgical scissor tips downward with slight pressure across the occipital bone beginning just between the pinnae.

Make a small 3- to 5-millimeter midline incision slightly above the palpated concavity. Draw 5 microliters of the cell suspension into a 30G Hamilton syringe. Use blunt-tipped forceps with a 1- to 2-millimeter tip to gently press down on the cisterna magna.

Introduce the tips in a closed position and open them while applying downward pressure on the dura. Repeat the blunt dissection until the dural membrane is easily identified and the associated blood vessels are visible in the exposed area.

While holding the forceps, open to retract surrounding musculature. Introduce a 30G non-coring needle under the dura to visualize the bevel, making sure that the needle is only introduced just beyond the bevel itself.

Slowly deploy a syringe plunger and deliver cells just below the dura. If leakage at the injection site is noted, apply gentle pressure with a cotton-tipped applicator. Close the skin by applying a wound clip or a microdrop of skin adhesive.

Monitor the mice daily after the surgery for the first week. If a mouse appears to be in pain or distress, treat it with a subcutaneous injection of 10 milligrams per kilogram carprofen, once every 12 to 24 hours for up to 5 days based on veterinary consultation and directive.