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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of CTCs

1. Calculate the number of CTCs needed for injection and prepare a single-cell suspension at 1.0 × 10⁴ cells/μL in sterile phosphate-buffered saline (PBS). Place the cell suspension on ice or at 4 °C throughout the procedure.
   NOTE: When using cell lines that require trypsinization, be sure to wash cells twice with sterile PBS to remove trypsin. Perform cell count using a hemocytometer or an automated cell counter. If a large cohort (>50 mice) is used, recount the cells and validate cell viability between injections to ensure a consistent number of cells are administered per mouse.

2. Presurgical Procedure

1. Inject the mouse subcutaneously with 5 mg/kg buprenorphine sustained-release (Bup-SR).
   NOTE: Do not inject the area of the proposed incision; choose an injection site well away from the scapula.
2. Anesthetize the mouse with 2–3% isoflurane until it shows no signs of the righting reflex. In addition, check for the tail and/or paw pinch reflex to confirm the state of anesthesia.
3. Prepare the mouse for surgery in a location remote from the operating area. Clip the surgical site (i.e., the dorsal surface of the skull) with enough border area to keep fur from contaminating the incision site; then, saturate the site with germicidal skin antiseptic, working from the center of the site to the periphery, and then allow to dry. Apply either sterile drape or a sterile adhesive-backed plastic drape material to protect the surgical site from contamination.
   NOTE: Instruments are to be autoclaved in advance and tips re-sterilized between animals using a glass bead sterilizer.
4. Shave the fur of the entire ventral surface of the head and prepare the skin using the sterile technique.
5. Situate the nose with a modified L-shaped nose cone of the stereotactic apparatus, ensuring the nares remain clear and open. Using tape across the ventral surfaces of both pinnae, gently pull the skin forward to secure it to the nose cone and bend the neck at an approximately 90° angle once secured. Administer 1.5% isoflurane to maintain anesthesia.
   NOTE: Proper positioning will result in the incision area being presented in a manner that allows for the easy identification of the caudal ridge of the occipital bone.
6. Position the body to ensure the spine is kept level with the cisterna magna, and while applying slight traction to the tail, place tape at the tail base to secure.

3. Surgical Cisterna Magna Injection

1. With the neck in full extension, and beginning just between the pinnae, run the surgical scissor tips downward with slight pressure across the occipital bone.
   NOTE: While in this midline position, a small depression is noticeable when the scissor tips dip into the concave area over the cisterna magna.
2. Make a small 3–5 mm midline incision just above the palpated concavity. Draw 5 μL of cell suspension at 1.0 × 10⁴ cells/μL (total 5.0 × 10⁴ cells) into a 30 G Hamilton syringe.
3. Use blunt-tipped forceps with 1–2 mm tips to gently press down on the cisterna magna. Introduce tips in a closed position and open them while applying downward pressure on the dura.
4. Repeat blunt dissection described in the previous step until the dural membrane is easily identified, and the associated blood vessels are visible in the exposed area.
   NOTE: The resulting injection window ensures blood vessels are undamaged during needle insertion.
5. While holding the forceps open to retract the surrounding musculature, introduce a 30 G non-coring needle under the dura to visualize the bevel. Ensure the needle is only introduced just beyond the bevel itself. Slowly deploy a syringe plunger and deliver cells just below the dura.
6. Properly place the bevel to observe the injection beneath the dura. Carefully administer the technique and use the 30 G non-coring needle to prevent damage to the membrane and ensure minimal leakage. If leakage is noted, apply gentle pressure with a cotton-tipped applicator.
7. Close the skin by applying a wound clip or micro-drop of skin adhesive. Once the injection is successfully performed, allow the mice to recover from anesthesia on a warm blanket, observing them continuously until they can maintain sternal position and demonstrate purposeful movement.
8. Monitor the mice daily after the surgery for the first week. If a mouse appears to be in pain or distress, treat it with 10 mg/kg subcutaneous injection of carprofen once every 12 to 24 h for up to 5 days based on veterinary consultation and directive. Allow mice to recover and monitor them for at least 48 to 72 h before proceeding to the next step.
   NOTE: Bup-SR, given preemptively, will last up to 72 h, so additional analgesics are not normally necessary. However, animals will be provided additional analgesics as necessary (if lethargic, not eating, ruffled). If a surgical site develops signs of postoperative infection (i.e., redness, swelling, tenderness, allodynia, hyperalgesia, hyperpathia, or suppuration), or if the mouse is guarding the affected area, euthanize the mouse. If cancer cells are successfully injected into the CSF, LMD and tumor progression will develop in the CNS within 1 or 2 weeks (depending on the types of CTCs or cell line used) (Figure 1).

Figure 1: Injection of circulating tumor cells into the cisterna magna in a murine xenograft model to study leptomeningeal disease and central nervous system metastases. (A) An illustration showing the location of the cisterna magna and the CSF accessing site, in which CTCs are injected using a Hamilton syringe. (B) A representative IVIS image of mice that had developed leptomeningeal disease and central nervous system metastases (brain and along the spinal cord) after 2 weeks of injection with circulating tumor cells. Cells were labeled with a luciferase reporter gene. Control animals injected with saline did not develop tumors (n = 3), and the experiment was performed in triplicate. Abbreviations: CTCs = circulating tumor cells; IVIS = in vivo imaging system.