We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.
Our surgical procedure enables specific gene editing of leptomeningeal cells to study their function in many physiological and pathological conditions such as neurodevelopment and the spreading of bacterial meningitis. To minimize damage during the intracisternal delivery of endoxifen, we utilize a bent needle that can be secured against the caudal edge of the skull to prevent it from penetrating deeper in the tissue. The procedure can be used to probe the role of genes expressing leptomeningeal cells through loss and gain of function experiments.
It can also be adopted to track the cerebrospinal fluid flow. Visual demonstration of this method will ensure successful identification of the injection site for intracisternal injection and understanding on how to secure the needle against the occipital bone. Begin by preparing a Hamilton syringe with a 30 gauge beveled needle for injection.
Use forceps to bend the needle to 30 degrees three millimeters from the tip. Next, dilute endoxifen in 10%DMSO to a concentration of one milligram per milliliter and backfill the syringe. Anesthetize the animal in the isoflurane chamber.
Then adjust the mouse head holder so that the mouthpiece is approximately at a 30 degree angle from the surface of the surgical table and fix the animal's head onto the holder. To improve accessibility to the cisterna magna, position the animal's body at approximately 30 degrees from the surface of the table with the head tilted downward which will establish an angle of 120 degrees with the rest of the body and extend the back of the neck to facilitate access to the cisterna magna. Once the animal is properly positioned, apply ophthalmic ointment, shave the back of its neck, and sanitize the area with alcohol wipes and Betadine.
Use surgical scissors to make a midline incision starting at the level of the occipital bone and extending posteriorly. Gently separate the superficial connective tissue and neck muscles by pulling sideways from the midline with fine tip tweezers which will expose the dural membrane overlying the cisterna magna. Position a small surgical separator to enable visualization of the cisterna magna throughout the procedure.
Identify the caudal end of the occipital bone and insert the previously bent needle immediately underneath. Once the dura has been perforated, allow the bent tip of the needle to penetrate underneath the surface by gently pulling the syringe upward and parallel to the animal's body which will ensure better stability. Inject the compound slowly to avoid interference with cerebrospinal fluid's natural flow.
After the injection, let the needle rest inside for one minute, then carefully remove it with the help of fine tip forceps. Close the skin with a few drops of cyanoacrylate adhesive and apply local anesthetic at the injection site. Remove the animal from the holder and place it in a clean cage on a heating pad.
Then make sure to monitor the animal until it regains consciousness. Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter, an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labeling the neighboring Cx30 expressing surface astrocytes and parenchymal astrocytes in the cortex. This injection protocol takes advantage of the physiological movement of the cerebrospinal fluid so the endoxifen solution is distributed throughout the subarachnoid space to efficiently recombine leptomeningeal cells overlaying the olfactory bulbs, cortex, and cerebellum.
The solution does not cross the brain meningeal barrier or come in contact with astroglial cells of the parenchyma as opposed to systemic administration through oral gavage. Recombination of leptomeningeal cells after intracisternal injection was identified through Pdgfr-alpha reactivity while surface and parenchymal astrocytes expressing Gfap remained unlabeled. When attempting this experiment, it is important to locate the injection site for intracisternal delivery.
This visual demonstration provides the technical know-how to carry out the procedure while minimizing the risk of damaging the neural tissue under it. Gene ablation studies can be performed with this technique to investigate the role of leptomeningeal cells in corticogenesis and regulation of cerebrospinal fluid formation and to identify additional sites for spreading of bacteria in the subarachnoid space.
