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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Subjects
- Use male Sprague-Dawley rats (250-300 g at arrival).
- House rats in solid-bottom cages in a colony room with a humidity of 40-60% and a room temperature (RT) of 21 ± 1 °C.
- Make water and food available at libitum.
- Keep rats under a reversed 12:12 hr dark/light cycle (lights on at 20 hr).
2. Surgery
- Per rat, prepare two pieces of chromic gut ligature (5-0) of approximately 6 cm long and place them in sterile saline to avoid drying and becoming stiff and brittle.
- Anesthetize the animal with pentobarbital (60 mg/kg, intraperitoneal [i.p.]) and treat with atropine (0.1 mg/kg, i.p.). Monitor the level of anesthesia by extending one leg and pinching the web of skin between the toes with a fingernail to ensure adequate pressure is applied. Ensure that the animal does not flex its limb. If necessary, administer additional pentobarbital.
- Shave the head of the rat so that a mid-line scalp incision can be made of approximately 25 mm with equal distances anterior and posterior to the center of the eyes.
- Fix the rat’s head in a stereotaxic frame or otherwise fixate the rat’s head. Place the rat on a heated pad to maintain body temperature.
- Apply ophthalmic ointment on both eyes to prevent damage from drying.
- Scrub the shaved head area with alcohol, then betadine.
- Make a mid-line scalp incision, exposing the skull and nasal bone.
- Use an operation microscope to perform steps 2.9 to 2.16.
- Dissect the edge of the orbit free, formed by the maxillary, frontal, lacrimal and zygomatic bones, using a cotton-tipped swab and a pair of Dumont forceps. Use the swab to soak up blood from possible bleeding.
- To give access to the infraorbital nerve (IoN), gently deflect the orbital contents with a precision cotton swab, taking care not to overstretch the anterior ethmoidal nerve which crosses superior to the IoN. Dissect the IoN free from the surrounding connective tissue, using a precision cotton-tipped swab and a pair of Dumont forceps using a spreading motion. Use the swab to soak up blood from possible bleeding.
- To perform a ligation, push one end of a single piece of chromic gut downward along the frontal bone, medial to the IoN and advance a few millimeters parallel and inferior to the IoN. The ligature is now held in place between the IoN and the frontal bone.
- Slip the tip of a 45-degree angled Dumont forceps under the IoN to place it in close proximity to the ligature. Gently retract the IoN laterally to reveal the ligature and tip of the forceps. Grip the ligature with the forceps and withdraw the forceps laterally from under the IoN.
- Pull out the ligature from the orbital cavity until both ends of the ligature are more or less equidistant from the IoN.
- Make a “slip knot” from the two ends of the ligature to allow perfect control over the degree of constriction and slide the knot against the IoN. Slide the knot further to constrict the IoN so that the diameter of the nerve is reduced by a just noticeable amount. Make a normal knot on top of the slip knot so that the amount of constriction remains constant (i.e., to prevent the slit knot from slipping).
- Cut the ligatures to leave approximately 2 mm of free ends from the knot.
- Repeat the same procedure (steps 2.11 to 2.15) to make a second ligation 2 mm apart from the first.
- For sham surgery, perform steps 2.2 to 2.10.
- Close the scalp incision using polyester sutures (4-0) and allow the rat to recover on a heated pad. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency and do not return the animal to the company of other animals until it is fully recovered.
- To avoid pre-emptive analgesic effects on the development of neuropathic pain, do not administer analgesia for post-surgical pain.