This protocol provides step-by-step guidelines for a reproducible route of access to regions of interest in the caudal brainstem and the upper cervical cord. This technique increases precision when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. This technique has been and can be applied to other animal models.
Dr.Veronique VanderHorst, an associate professor of neurology and principal investigator of the VanderHorst Laboratory, will demonstrate this procedure. Make sure that the oxygen flow is directed to the nose cone. Move the mouse to the stereotaxic frame and place the nose in a flexible nose cone.
Place the mouse in the stereotaxic frame using ear bars only. Place lubricant on the eyes. Anteroflex the mouse's head to a 90 degree angle by manually guiding the nose.
To secure this position, place a plastic barrier between the ear bar pillars of the mouse adapter parallel to the pillars with the flat part of the skull serving as the reference. Place the heating pad underneath the mouse and make sure that the neck and the rest of the body are positioned parallel to the table by elevating the body with a small box. Place a drape underneath the body.
Inject a single dose of four milligrams per kilogram Meloxicam slow release subcutaneously at a volume of two microliters per gram of body weight. Clean the surgical incision site first with a 70%alcohol prep pad, then with a Betaine prep pad, and then again with an alcohol prep pad and let it dry. Disinfect hands and put on sterile gloves, then place a drape at the surgical site.
Ensure that the mouse is appropriately anesthetized by pinching the toes or checking the corneal reflex. Reduce isoflurane to maintain the levels at 2.0. Make a one to 1.2 centimeter incision with a surgical 10 blade from the edge of the occipital bone toward the shoulders in one smooth movement.
Carefully make an incision in the midline raphe of the trapezius muscle, exposing the paired longus capitis muscles. Place both retractor hooks between the paired longus capitis muscles, one oriented to the left and the other to the right. Use the blunt laminectomy forceps to separate the left and right bellies of the paired longus capitis muscle, starting from the occiput where the midline is readily visible.
Guide the blunt forceps across the bone of the occiput in the midline down to where it meets the cisternal dura mater and then continue across the dura mater to the atlas. Reposition the retractors and adjust the tension by repositioning the hemostats. Use the blunt laminectomy forceps to separate the muscles further in the midline to obtain a good view of the brainstem and cerebellum.
Repeat the above procedure as needed until the cerebellum and brainstem appear below the dura. Using blunt laminectomy forceps, clear the dura of the small strands of connective tissue by moving the forceps from the midline in a lateral direction until there is a clear view of the brainstem, creating more lateral space. View the dorsal surface of the brainstem with detailed landmarks through the open dura.
Use the angled forceps to grab the dura extending from the occipital bone to the atlas, then use the spring scissors to make a small opening of approximately 0.5 to 1.5 millimeters in the dura. Once the dura is opened, drain excess cerebrospinal fluid with a sterile Q-tip. The obex, the point where the central canal opens into the fourth ventricle, is the standard anterior-posterior and mediolateral zero point.
Position the pipette or syringe to the target using the stereotaxic arm. Lower the arm of the dorsal onto the dorsal surface which forms from the dorsoventral zero point. Then lower the pipette onto the brainstem and inject the solution.
Leave the needle in place for one to five minutes after injection to avoid a needle track when using volumes between three to 50 nanoliters. Then lift the pipette or syringe using the stereotaxic arm and repeat this for multiple targets. Remove the hooks carefully from the surgical field.
The paired longus capitis muscles will fall back into a neutral position fully covering the cisterna magna. Do not close the trapezius muscle and dura mater in the midline as they are too fragile to hold sutures. Close the skin with 3 nylon or polypropylene sutures.
The cisterna magna approach makes it possible to target the caudal brainstem and upper cervical cord structures that are otherwise hard to reach via standard stereotaxic approaches or are prone to inconsistent targeting. In mice, structures such as the hypoglossal nucleus, ventral respiratory group and adjacent reticular formation in the caudal brainstem have been routinely targeted using the cisterna magna approach as illustrated here for the hypoglossal nucleus and the ventromedial medulla. In order to determine the accuracy of the cisterna magna approach versus the standard approach, the distance between the intended and actual target sites in the anteroposterior, mediolateral, and dorsoventral planes for ventral and dorsal regions was measured.
The results show significantly smaller errors in the anteroposterior, mediolateral, and dorsoventral planes compared to the standard approach, highlighting the enhanced accuracy of the cisterna magna approach for these targets. When attempting this protocol, it is critical to make sure that the anteroflexion of the head and the elevation of the body is performed as described. Next, it is important to recognize key landmarks before manipulating the muscle or the dura mater.
If these landmarks are not recognized or if they're lost, it will be challenging to stay oriented and execute the procedure as intended. This technique helped address conceptual questions related to the functional anatomical organization within the caudal brainstem and upper cervical cord.
