Fluorescence-guided Tumor Resection and MSC-seeded Scaffold Implantation in Murine Model: A Surgical Procedure to Deliver Therapeutic Mesenchymal Stem Cells into Brain Tumor Resection Cavity in Mouse Model

Protocole

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

1. Fluorescence-guided Resection and Scaffold Implantation

NOTE: Sterilize all tools prior to initializing surgery.

1. Place the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope.
2. Anesthetize the mouse under inhaled isoflurane in an induction chamber. Once anesthetized, secure the mouse in the stereotaxic frame with continuous inhaled anesthesia supply via an isoflurane nose cone adapter. Maintain body temperature with a heating pad and/or probe.
   NOTE: 4–5% isoflurane is suitable for induction and 2–3% is suitable for maintenance, but this must be adapted and monitored for each mouse.
3. Apply ophthalmic ointment to the eyes to prevent drying of the cornea. Sterilize the incision site of the scalp with a series of three alcohol and three betadine wipes.
4. Perform toe pinch reflex test on each limb and confirm negative response to ensure proper anesthetization. Ensure a steady respiratory rate at approximately 55–65 breaths/min.
5. Using forceps, pinch and gently lift the scalp. Make a midline linear rostral-caudal incision with surgical scissors. Irrigate the incision site with PBS and remove subdermal fat with a cotton tip applicator in a circular brushing motion. Arrange the skin such that the previously-established cranial window is fully visible.
6. Gently puncture the dura just interior to the borders of the cranial window using an 18G needle. Repeat until the incision fully traces the interior of the window.
7. Remove the dura by peeling it away using fine forceps, revealing the underlying parenchyma and tumor.
8. Locate the U87 mCh-Fl tumor by turning room lights off and turning the stereomicroscope fluorescence-mode on. Prepare for resection by loading a 1–200 µL pipette tip into the end of the tubing of a vacuum pump.
9. Turn the vacuum pump on. Resect the tumor by gently aspirating fluorescent tissue until no signal remains. Turn the vacuum pump off and discard the pipette tip. Turn the fluorescence off and the room lights back on.        
   NOTE: To control bleeding, irrigate with cold PBS and apply steady pressure with a cotton tip applicator. In more severe cases, the resection cavity can also be temporarily packed with a hemostatic agent as long as it is removed after the bleeding has stopped followed by another PBS irrigation.
10. Immediately prior to implantation, slowly dip an MSC-seeded PLA scaffold in PBS to remove unwanted media and associated components. Implant the scaffold into the resection cavity. If needed, add 1 µL fibrinogen (67–106 mg/mL) followed by 1 µL thrombin (400–625 U/mL) to secure the scaffold in place.
11. With the dura removed and the bone flap from the cranial window already discarded, seal the wound simply by closing the skin and applying surgical glue. Remove the animal from inhaled anesthetic and allow to recover on a heated surface.

Access restricted. Please log in or start a trial to view this content.

matériels

Name	Company	Catalog Number	Comments
Just for mouse stereotaxic instrument	Stoelting	51730	Maintains steady head positioning during surgery
Fluorescence dissecting stereomicroscope	Leica	M165 FC	Allows real-time imaging of tumor during resection
Vetbond tissue adhesive	3M	1469	Sugical glue to close skin wound
Artificial tears	Akorn	664268	Prevents eyes from drying during surgery
Webcol alcohol preps	Covidien	6818	Sterilize incision site
Betadine surgical scrub	Purdue Fredick Company	6761815117	Sterilize incision site
Cotton-tipped applicators	Fisherbrand	23-400-115	Surgery tool
E-vac aspirating system	Argos	EV310	Vacuum pump used to resect tumor
Fibrinogen and thrombin extracted from as-received TISSEEL	Baxter		To temporarily secure the scaffold in the resection cavity