This article discusses the glymphatic system in the vertebrate brain, which facilitates fluid exchange similar to the lymphatic system. The study outlines a method for visualizing cerebrospinal fluid distribution using fluorescent tracer molecules.
The vertebrate brain contains a specialized fluid exchange system analogous to the lymphatic system, called the glymphatic system. In this system, cerebrospinal fluid or CSF enters the periarterial spaces, allowing solute exchange via specialized channels present in the astrocytic endfeet. The solute-carrying CSF flows into the perivenous space to flush the solutes from the brain. This pathway is significant for brain physiology and can be visualized using fluorescent tracer molecules.
To begin, prepare an anesthetized pig in the prone position with its head flexed upward. Make an incision below the occipital crest. Retract the muscles to expose the cisterna magna - an opening in the subarachnoid space between the cerebellum and medulla oblongata.
Carefully insert a cannula through the dura mater into the cisterna magna. Apply dental glue around the cannula. Allow it to harden. Thereafter, inject fluorescent tracer molecules suspended in artificial CSF at an appropriate rate. A visibly pulsating tracer solution at the cannula’s base confirms successful delivery.
Following injection, reposition the tissue layers and suture the incision. Place a wet cotton gauze over the incision to limit heat loss. Allow the tracer molecules to circulate for the desired period. When visualized under a fluorescence microscope, tracer molecules help visualize the CSF distribution in the glymphatic system.
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 superglue 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.
繁體中文
