Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The overall goal of this surgical technique is to deliver exogenous substances into the mouse brain without penetrating the cranium. This method can help answer key questions in neuroscience regarding the action of drugs, pathogens, and exosomes in the brain. The main advantage of this technique is that it causes minimal trauma to the animal, preserves the integrity of the neuronal network, and achieves high concentrations of the substance in the brain.

Begin by administering isoflurane mixed with oxygen to anesthetize the mouse in an induction chamber. After induction of anesthesia, transfer the animal to the surgical surface under a stereo microscope, and place the nose cone to maintain a surgical plane of anesthesia. Ensure that the respiratory rate is around one to two respirations per second without gasping.

In addition, ensure that the animal does not react to whisker stimulation or a toe pinch. Use a sterile swab to apply a drop of ophthalmic lubricant on each eye to prevent dryness. After shaving the surgical site with a razor, prep the area with 70%ethanol and chlorhexidine.

While looking through the stereo microscope, use surgical scissors and forceps to create a midline incision in the neck from above the breastbone to below the jaw. Use forceps to carefully separate fatty and connective tissue to expose the trachea. Then place a soft, round object of about zero point five centimeters in diameter under the back of the neck to extend the neck and further expose the area.

Next, separate the tissue around the trachea using hooks or a tissue retractor. On the left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery, or CCA. Using forceps, carefully remove all connective tissue to expose the CCA bifurcation in the beginning of both the external and internal carotid arteries.

Once the ECA and ICA are exposed, use forceps to place a segment of one-centimeter nylon suture under the external carotid artery. Then place a second one-centimeter segment. Then tie a permanent knot at the highest point possible of the ECA.

At the lowest point possible of the ECA, immediately above the CCA bifurcation, tie a looser, removable knot. Then use vessel clips to close the CCA at the lowest possible point, and to close the ICA. Properly closing the internal carotid artery before making a small cut in the external carotid artery will prevent bleeding.

Now, using microdissection spring scissors, make a two-millimeter cut in the ECA between the two knots. It is important to keep the cuts small, as a large cut will cause leaking of the substance to be infused. Assemble the infusion system by attaching six inches of capillary tubing to a tuberculin syringe containing 250 microliters of the substance to be infused.

Then gently insert the tip of the capillary tubing into the incision in the ECA. Keep advancing the tip until it reaches midway between the bifurcation and the clip on the CCA. Tie down the lower ECA knot.

Verify that the knot is loose enough to allow capillary fluidity, and tight enough to prevent leaking. Then, remove the clip from the ICA. Gently apply pressure to the syringe piston, allowing substance infusion at approximately 10 microliters per second.

Following infusion of the test substance, place the clip back on the ICA. Gently remove the capillary tubing. Then, tie down the lower ECA knot completely.

Remove the clip from the ICA and the CCA. Then remove the retractor or tissue hooks and pillow. Clean the area to be sutured with sterile saline, and then close the incision using nylon sutures.

After administering an approved anti-inflammatory agent and analgesic to alleviate post-operative discomfort, place the animal in a cage on a heating pad for at least one hour, and monitor recovery. This histogram demonstrates HIV detection by real-time PCRs seven days after viral infusion via the left internal carotid artery. As expected, levels of the virus are higher in the left hemisphere.

This immunofluorescence image shows a mouse brain section depicting GFP tag CD63, an exosome marker, after extracellular vesicle infusion via the internal carotid artery. The CD63 expressing extracellular vesicles, shown in green, are associated with the mouse brain microvessel. Once mastered, this technique can be done in 20 minutes if it is performed properly.

After its development, this technique paved the way for researchers in the field of neurodegenerative diseases to explore drug, pathogens, and exosome interactions in mice.