This protocol is important as it allows for investigation of the glymphatic system in a large mammal, thereby bringing us closer to understanding the glymphatic system in humans. There are two advantages to this technique. Introduction of traces into the cisterna magna avoids any direct damage to the brain, which is in contrast to intraventricular injections.
Secondly, this is a direct approach which allows immediate visualization of the cannulation and knowing whether or not it has been successful. Begin by placing the pig in a prone position. Palpate the back of its head and neck to locate and mark the occipital crest, the spine of the first thoracic vertebrae, and the base of each ear.
Draw a straight line between the crest and the vertebrae along the longitudinal axis. Then following the base of the skull, draw two lines from the crest to the base of each ear. Carefully clamp the animal's tail and watch for a reflex to see if it is in a deep sleep.
Begin by making a dermal incision along the longitudinal line down to the muscle using a scalpel with a number 21 blade, then extend two perpendicular incisions further along the shoulders, each 10 to 15 centimeters in length. Starting from the occipital crest, make dermal incisions along the line down to the base of each ear. Use anatomical forceps to grip the skin corners formed at the occipital crest, then run the scalpel blade lightly over the fascia to separate the skin from the underlying muscle.
Resect the skin along each of the five incisions to visualize parts of the trapezius muscle. Use the scalpel to make a longitudinal incision approximately one centimeter deep where the trapezius comes together at the midline, then perform a blunt dissection along the longitudinal cut in the muscles using a combination of straight and curved surgical forceps, which will separate the bellies of the trapezius and underlying semispinalis capitis biventer muscle. Sever any persisting muscle fibers with a scalpel and continue to perform blunt dissection until the semispinalis capitis complexus becomes visible.
Move along the posterior aspect of the skull and sever the origins of the trapezius and semispinalis capitis biventer muscles. To separate the two muscles longitudinally, use the surgical forceps to perform blunt dissection until the semispinalis capitis complexus is fully visible. Then use the self-retaining retractors to retract the trapezius and semispinalis capitis biventer muscles.
Use the scalpel to make a one centimeter deep longitudinal incision where the bellies of the semispinalis capitis complexus come together at the midline. Perform a blunt dissection using surgical forceps working along the longitudinal cut between the muscle bellies until both the atlas and axis are palpable. Move along the posterior aspect of the skull, severing the origins of the semispinalis capitis complexus muscles.
Use the scalpel and blunt dissection to separate the muscle longitudinally from the underlying vertebrae, then use another set of self-retaining retractors to retract the semispinalis capitis complexus muscles. Use a scalpel to carefully remove any remaining tissue overlying the region where the atlas meets the skull base. Place one arm onto the animal's neck and one finger at the juncture of the atlas and the skull, then simultaneously elevate the head and flex the neck while palpating with the finger to reveal the cisterna magna.
Ensure that one person elevates and flexes the head and neck of the animal while the other palpates for the cisterna magna, making a note of its anatomical location. Introduce a 22 gauge cannula slowly and carefully into the cisterna magna through the dura at an angle oblique to the longitudinal axis, then retract the needle from the cannula and place a cap on the lock. Start with applying super glue and an accelerator where the cannula enters the tissue, then apply the dental cement.
Wait five minutes for the cement to harden. Remove the cap carefully from the cannula. Attach the cannula to the male end of the IV line tap with the tracer using a 10 centimeter extension.
Inject the tracer slowly at a rate of 100 microliters per minute either by hand or using a micro infusion pump. Remove the IV line tap and replace it with the cap. Check if the tracer is visible pulsating at the base of the cannula.
Then place sandbags under the neck of the animal to maintain some flexion. Release the head and leave the animal in a resting prone position. Release the self-retaining retractors and replace the muscles.
Use the surgical towel clamps to bring the skin together over the muscles. First use gauze and then a blanket to cover the towel clamps and the incision to limit heat loss. Allow the tracer to circulate for the desired amount of time.
Stitched macroscopic images of the brain's dorsal surface can provide detailed insights into the distribution patterns of the tracer across the sulci and fissures. Similar images from the brain's ventral and lateral surfaces can provide information about the tracer distribution in the temporal lobe and the lateral fissure. Images of the brain surface of higher magnification produced using a stereoscope can help visualize the tracer in the PVS along the arteries.
Macroscopic coronal brain sections provide insight into the depth of tracer penetration in the interhemispheric fissure and subcortical tracer distribution and structure, such as the hippocampus and striatum. Immunohistochemical staining for AQP4, glial fibrillary acidic protein, and smooth muscle actin showed that the tracer localized within the PVS, as well as moved into the brain parenchyma. Astrocyte foot processes that form the outer surface of the PVS are identified using AQP4 and glial fibrillary acidic protein staining.
The endothelial cells that form the inner surface of the PVS are stained using lectin and GLUT-1 stain. SMA staining identifies arteries and arterioles and can be used to show that PVS influx occurs along the arteries instead of veins, which constitutes the basic physiology of normal glymphatic function. Going forward, we hope to explore the glymphatic system at a high resolution in large mammal in the context of neuropathology such as stroke and traumatic brain injury.
