Targeted Substance Delivery to the Mouse Brain via Infusion into the Internal Carotid Artery

Samenvatting

Source: Leda, A. R., et al. Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery. J. Vis. Exp. (2017)

This video demonstrates a microsurgical procedure designed to facilitate the targeted infusion of a test substance into the brain via the internal carotid artery (ICA). The procedure involves precise suturing and vascular manipulation to temporarily restrict blood flow, ensuring controlled delivery. A capillary tube is inserted into the external carotid artery (ECA) and advanced toward the bifurcation. Upon infusion, the substance bypasses systemic circulation, crosses the blood-brain barrier, and achieves targeted delivery to the brain parenchyma.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Mice for Surgery

1. Anesthetize mouse with isoflurane mixed with oxygen, using a laboratory anesthesia system. Use isoflurane at setting between 4-5% and oxygen flow at 2 L/min on the commercial machine (See Materials Table). Transfer the animal to the surgery surface, under a stereomicroscope, and sustain anesthesia using a nose cone (use isoflurane setting 1.5 - 2.5 and oxygen flow at 2 L/min).
   1. Ensure that the mouse respiratory rate is around 1 - 2 respiration/sec without gasping. In addition, ensure that the animal does not exhibit whiskers stimulation reaction and pedal reflex (toe pinch). Monitor respiration rate and effort during the surgery, at least every 5 min. Follow specific Institutional Animal Care and Use Committee and veterinary guidelines for rodent anesthesia monitoring.
2. Apply a drop of ophthalmic lubricant on each eye using a sterile swab in order to prevent dryness. Administration of anti-inflammatory and analgesic to alleviate discomfort is recommended.
3. With the animal lying on its back, maintain anesthesia using a nose cone.
4. Clean the animal's neck area by wiping the area three times with ethanol 70% and chlorhexidine. Shave the animal's surgery area using a razor (detailed below).

2. Dissection of the Common Carotid Artery (CCA)

1. Perform the entire microsurgery procedure under a stereomicroscope. Using surgical scissors and forceps perform a shallow midline incision in the neck, from above the breastbone to below the jaw (about 3 to 4 cm).
2. Using forceps carefully separate fatty and connective tissue to expose the trachea.
3. Place a pillow (round object, about 0.5 cm in diameter) on the back of the neck of the mouse to extend the neck, further exposing the area.
4. Separate the tissue using either a tissue retractor or hooks.
5. On the animal's left side of the trachea, carefully tweeze apart the connective tissue to expose the left CCA.
6. Using forceps carefully remove all connective tissue to expose the CCA bifurcation, and the beginning of both external and internal carotid arteries.

3. Preparation of the CCA for Substance Infusion

1. Insert two segments of nylon suture (about 1 cm each) under external carotid artery (ECA), using forceps.
2. Place a permanent knot at the highest point possible of the ECA.
3. At the lowest point possible of the ECA, immediately above the CCA bifurcation, place a removable knot. This knot should be loose compared to the upper knot.
4. Close the CCA using a vessel clip, at the lowest possible point.
5. Close the internal carotid artery (ICA) using a vessel clip.
6. Using microdissection spring scissors perform a small cut in the ECA (about 2 mm), between the two knots.

4. Substance Infusion via ICA

1. Assemble an infusion system. Attach 6 inches of capillary tubing (ideal dimensions: 2.5 mm x 1.2 mm) to a tuberculin syringe containing 250 µl of the substance to be infused (drugs, pathogens, extracellular vesicles, among others) and insert the capillary tip gently into the incision performed in step 3.6.
2. Continue to slide down the capillary until it reaches a midpoint between the bifurcation and the clip closing the CCA.
3. Tie down the lower ECA knot. Verify that the knot is loose enough to allow capillary fluidity and tight enough to prevent leaking.
4. Remove clip from the ICA.
5. Gently apply pressure to the syringe piston allowing substance infusion at approximately 10 µl per second.

5. Post Infusion Procedures

1. Place clip back on the ICA.
2. Gently remove the capillary tubing.
3. Tie down the lower ECA knot completely.
4. Remove clips from the ICA and the CCA.

6. Incision Closing and Post-operative Care

1. Remove the retractor or tissue hooks and pillow.
2. Clean suture area using sterile saline.
3. Close the incision using nylon suture/needle and forceps.
4. Administer anti-inflammatory and analgesic to alleviate post-operative discomfort.
5. Place the animal in cage placed on heating pad for at least 1 hr and monitor recovery.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Anesthesia instrument	Vetequip	901806
Surgical scissors	Fine Science Tool	14558-09
Surgical forceps straight tip	Fine Science Tool	00108-11
Surgical forceps angled tip	Fine Science Tool	00109-11
Spring scissors	Fine Science Tool	15000-08
Nylon suture	Braintree Scientific	SUT-S 104
Capillary tubing (Micro-Renathane 0.010” x 0.005” per ft.)	Braintree Scientific	MRE01050
Closing suture	VWR	95057-036
Isoflurane	Piramal
2,3,5-Triphenyltetrazolium chloride	FisherScientific	50-121-8005