Generating a Rat Model of Tibial Neuroma Transposition

Samenvatting

Source: Brakkee, E. M., et al. Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats. J. Vis. Exp. (2023).

This video demonstrates a procedure to generate a rat tibial neuroma transposition (TNT) model. After exposing the tibial nerve in the hind leg of an anesthetized rat, transect the nerve. Next, create a subcutaneous tunnel, thread the severed nerve through the tunnel, and suture it in place. In the absence of a target for reinnervation, disorganized axonal outgrowths from the severed nerve form a neuroma, which triggers neuropathic pain by exciting nociceptors.

Protocol

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

1. Anesthesia and preparation

NOTE: This study was conducted on 15 male Sprague Dawley rats that were 12 weeks old.

1. Anesthetize the animals by induction with 5% isoflurane and maintain anesthesia with 2%-3% isoflurane.
   NOTE: Maintenance with 2% isoflurane usually results in sufficient anesthesia and spontaneous breathing, without the need for tracheal intubation or mechanical ventilation.
2. Check the animals' reflexes by pinching the foot with tweezers. Be sure that the animal is unresponsive before proceeding. Shave the operative field from knee to ankle with an electric shaver and apply ophthalmic ointment to the eyes to prevent dryness. Inject 0.5 mg/kg of analgesic carprofen subcutaneously in the abdominal region.
3. Place the anesthetized rat on its back with its head either to the left or right and the leg to be operated on near the surgeon. Exorotate the lower hind limb so that the medial malleolus faces upward. Place the rat under a stereo surgical microscope with 6x magnification.
4. Disinfect the shaved area with three alternating rounds of iodine-based scrub followed by alcohol. Place a sterile sheet with an operative hole over the leg, so only the operative field is visible. Ensure to maintain these sterile conditions during the surgery.

2. Surgery

1. Place a small cotton swab below the ankle in order to keep the operative field horizontal. Locate the knee and gently make a longitudinal incision of 1-2 cm using a scalpel over the medial side of the hind paw from mid-calf to ankle. If needed, open the skin and subcutis further with micro scissors until the muscle layers are visible.
2. Identify the superficial neurovascular bundle as two or three white and a thicker purple/red line, sometimes with minor branches, that can move freely over the muscle layers. Using an electrocautery (see Table of Materials), coagulate any active bleeding or oozing in the operative field. Be careful not to damage the neurovascular bundle.
3. Bluntly dissect to open the fascia in between the gastrocnemius muscles, just posterior to the superficial neurovascular bundle of 2.2. Between the fascia of the muscles, the tibial nerve can be found. The tibial nerve is about three times the size of the superficial nerve in the neurovascular bundle. Use the tibial bone as an additional landmark (the tibial nerve lies just posterior to the tibial bone).
4. Identify the tibial nerve and its bifurcation.
   NOTE: The bifurcation is usually visible with a lighter line longitudinally over the nerve.
5. Carefully dissect the tibial nerve free from the surrounding vascular bundles. Perform the dissection by bluntly moving the tibial nerve and cutting the exposed tissue that shows some stretch while moving the tibial nerve.
   NOTE: If the tibial nerve is attached to crossing veins after dissection, these veins can be coagulated in order to expose the whole tibial nerve. Be careful not to coagulate the tibial bundle itself.
6. Expose the tibial nerve proximally until it disappears underneath a crossing muscle layer. At this point, the tibial nerve seems to dive more deeply into the hind paw toward the knee. Expose the tibial nerve distally up to the ankle.
   NOTE: When the tibial nerve is exposed more distally, the amount of crossing collagen fibers (i.e., fibers perpendicular to the direction of the nerve fibers) will increase. These collagen fibers need to be cut to enable enough length for transposing the tibial nerve.
   1. When the whole tibial nerve is exposed, place the muscle layers back to avoid dehydration of the nerve. If the nerve dehydrates (i.e., it becomes more stiff, dull, and wrinkly), and covering with muscle layers does not suffice, add drops of saline to hydrate it.
7. Using a blunt microsurgery tool, preferably a micro needle holder, dissect the pretibial skin from the subcutaneous muscle layer in order to make a subcutaneous tunnel. To do this, hold the skin up and push the blunt tip into the tissue, parallel to the skin. Make sure the end of the tunnel is located pretibially or more laterally to ensure easy accessibility of the area for testing the neuroma.
8. Increase the isoflurane to 5%. Return to the tibial nerve and expose it (i.e., go back to the place described in step 2.6). Cut the tibial nerve (i.e., both plantar branches) at the most distal level near the ankle. Decrease the isoflurane to the normal level of 2%-3%.
9. Change the microscope magnification to 10x or 16x. Identify the epineurium of the tibial nerve proximal to the cut made in step 2.8, or in case of a more proximal bifurcation of the tibial nerve, identify the epineurium of both the medial and lateral plantar branches proximal to the cut in step 2.8.
   NOTE: The epineurium is whiter and firmer compared to the nerve fibers within, which are more yellow and soft.
10. Carefully place a 8-0 nylon suture (see Table of Materials) through the epineurium of the proximal nerve end by carefully holding the epineurium with tweezers and putting the needle between the nerve and epineurium with a bite of approximately 0.5 mm. Pull the suture through and take a bite with the needle subcutaneously at the end of the subcutaneous tunnel made in step 2.7. Make a knot, which will transpose the nerve laterally into the subcutaneous tunnel.
    NOTE: If both plantar branches share a common epineurium, one suture should suffice. If both plantar branches have their own epineurium, each epineurium should be fixated individually. Avoid placing the suture through the skin; only fix it subcutaneously.
11. Place a thicker suture with a dark color (preferably a blue or black 4-0 suture) flush to the fixated nerve end, not penetrating the skin. Make sure the suture is visible from the outside of the skin. Check whether the nerve stays in place after moving the paw and muscles. Cut off the suture ends with a slightly longer suture end on the 4-0 than on the 8-0 suture.
12. Change the magnification of the microscope back to 6x. Close the skin with intraepidermal sutures using the 8-0 suture and gently clean the skin with 0.9% NaCl using a cotton swab.

3. Post-surgical treatment

1. Place the rat in a clean cage under a paper towel in a comfortable position. If the room is cold, place a heat pad under a part of the cage (only under a part of the cage as the animal should be able to escape the heat when needed). Ensure easy access to food and water.
2. Do not leave the post-surgical rat unattended until it has regained sufficient consciousness to maintain sternal recumbency. The rat can be returned to the company of other animals when it has fully recovered from anesthesia after surgery. This is usually after 1 h and when the rat exhibits its normal walking pattern and behavior.
3. At 24 h and 48 h after the surgery, administer a dose of 0.5 mg/kg carprofen subcutaneously (abdominal region) to treat post-surgical pain.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Carprofen	Local Veterinary Pharmacy	n/a	The local veterinary pharmacy makes caprofen dilution
Cotton swabs	Nobamed	974255
Electrocautery	Fine Science Tools	18010-00
Ethanol 70%	Interchema BV	400406
Ethilon 4.0	Johnson & Johnson	1854G	IMPORTANT: the color should be blue or black
Ethilon 8.0	Johnson & Johnson	BV130-5
Isoflo, isoflurane Zoetis	Dechra Veterinary Products	B506
Mesh bottom cages	StoeltingCo	57816 and 57824
Micro forceps	Fine Science Tools	11251-35
Micro needle holder	Fine Science Tools	12076-12
Micro scissors	Fine Science Tools	15019-10
Micro tweezers	Fine Science Tools	11254-20
NaCl 0.9%	Trademed	H7 1000-FRE
Needle holder	Fine Science Tools	12004-16
Ophthalmic ointment	Local Veterinary Pharmacy	n/a	The local veterinary pharmacy makes the ophthalmic ointment
Scalpel	Fine Science Tools	10003-12
Scissors	Fine Science Tools	14001-12
Stereo surgical microscope	Leica	A60 F
Sterile sheet with hole	Evercare OneMed	1555-01
Surgical blade nr.15	Fine Science Tools	10015-00
Tweezers	Fine Science Tools	11617-12