The overall goal of this procedure is to study trigeminal neuropathic pain in rats via a chronic constriction injury to the infraorbital nerve. This is accomplished by first making a midline scalp incision and exposing the infraorbital nerve. The second step is to constrict the infraorbital nerve by placing two loosely tied ligatures around the nerve.
Next, the animal is placed in an observation cage for 10 minutes, and spontaneous neuropathic pain is assessed by measuring the amount of isolated face grooming. Additionally, mechanical allodynia is measured by stimulating the rat's infraorbital nerve territory with a graded series of Von Frey hair filaments ultimately changes in grooming behavior and response. In the EPS infraorbital nerve territory are used to investigate the treatment of trigeminal neuropathic pain.
The main advantage of the red Infraorbital nerve model over others like the sciatic nerve or spinal nerve ligation model is that the infraorbital nerve is a purely sensory nerve without a motor or autonomic components. Also, this model includes the measurement of spontaneous pain, which is the most important complaints in neuropathic pain.Patients. Animals are treated and cared for according to the guidelines of the Committee.
For Research and Ethical Issues of IASP, the protocol is approved by the Institutional Ethical Committee. In preparation, cut two six centimeter pieces of five zero chromic gut ligature, store them in a dish of sterile saline so that they do not stiffen up. After confirming that the rat is anesthetized by the absence of a reflex response, begin preparing it for surgery by shaving its head.
For a mid scalp incision, shave a 25 millimeter radius from the center point of the eyes. Next, fix the rat's head in a stereotaxic frame and put a heated pad under the rat's body. Then apply some ophthalmic ointment and clean the shaved area with alternating alcohol and Betadine scrubs.
Begin the surgery with a midline scalp incision to expose the skull and nasal bone. Proceed with the surgery using a dissection microscope. Next, using forceps free the edge of the orbit formed from the maxillary frontal lacrimal and zygomatic bones also swab up blood as needed.
Now to access the ION gently deflect the orbital contents with a precision cotton swab. In doing this, do not overstretch the anterior ethmoidal nerve, which crosses superiorly to the ION. Next, with the help of forceps, use a spreading motion to dissect the ION free from the surrounding connective tissue, always swab a bleeding as needed.
Now placed a piece of ligature between the ION and frontal bone by pushing one n downwards along the frontal bone, medial to the ION, and then advancing it a few millimeters parallel to the ION staying inferior to the ION. Then slip the tip of a 45 degree angled forceps under the ION close to the ligature and gently retract the ION laterally revealing the ligature and tips of forceps. Grab hold of the ligature and pass it under the ION, pulling it out until the exposed lengths are even.
Then tie the ends with a slip knot and slide the knot onto the ION visibly. Constrict the nerve just slightly and complete the knot so it can't slip any further. Now, cut off the free ends of the ligature and place another ligature using the same technique, two millimeters away from the first ligature.
Visually confirm the correct placement of the ligatures. Complete the surgery by closing the scalp with four zero polyester suture. Then monitor the rat as it recovers on a heated pad.
When it has regained sternal incumbency, return it to a proper cage. The rat should be housed alone until it has completely recovered. This test should be conducted in a dark room illuminated by a single 60 watt red light bulb.
One meter from the test site. Make sure there is sufficient background noise if necessary. Provide white noise at the test site.
Position a mirror along one side of the observation cage and position a video camera to view the rat and mirror on the opposite side. A clean test cage should always be used for each trial. Bring a single rat from the colony in an otherwise empty cage to the test site.
Put the animal in the test cage and for 10 minutes, record its behavior. After making the recording, return the rat to the colony and clean the test cage. After habituating the rats to the test site for a few days, analyze the subsequent data, watch the recordings, and score the grooming episodes.
Facial grooming is defined as movement patterns where the four paws contact the facial area. Distinguished isolated facial grooming from facial grooming preceded by or followed by grooming the body.Body. Grooming is any movement where the paws, tongue or incisors are brought in contact with a body area other than the face or the four paws.
Next record. The amount of time spent facial grooming and the number of grooming episodes. Grooming episodes are separated by at least four seconds of non-fatal grooming behavior.
Body grooming does not count as facial grooming, and two episodes of facial grooming can be interrupted by four or more seconds of body grooming. Transport the rats in groups of six to the test site in covered cages for the test. Transfer a single rat to a plastic cage with bedding and a hinged lid For easy access.
For the test, have five different Von Frey hairs. Apply the Von Frey hairs progressively from most flexible to most stiff for 10 minutes. Let the rat settle in the cage during this period.
Every 30 seconds, open the lid and gently touch a probing rod to the wall. After the habitation period when the rat has settled down and is just sniffing about, poke it with the first Von Frey hair within the ION Territory central to the SLE pad on the hairs skin surrounding the mesial vibra breast. The filament hard enough so it bends over about half a second.
Score the response of the animal for no response. Record a zero score one. If the rat notices the stimulus, but does nothing else score the response as two.
If the rat slowly withdraws from the stimulus, a face wipe counts as a two. Score, the response as three. If the rat attempts to attack or quickly withdraws the stimulus, if the rat responds by grooming asymmetrically with at least three face wash strokes to the stimulated region, then score the response as a four.
Typically six rats are tested simultaneously using one hair on each before the next hair is used. Once a hair has been applied to one side, apply the same hair to the contralateral side. When both sides have been stimulated, move on to the next hair.
For each rat, randomly choose which side to stimulate first. After the ION constriction operation, observation of the rats revealed that they spend significantly more time in isolated facial grooming. Ipsilateral mechanical stimulation of the ION territory resulted in nearly no response during the first operative week.
In the second postoperative week, the rats became hypersensitive. This persisted for up to 120 days. A very small increase in sensitivity on the contralateral side was also noted.
After watching this video, you should have a good understanding of how to expose and ligate the infraorbital nerve in the rat and how to perform behavioral testing that allows you to measure spontaneous neuropathic pain and mechanical allodynia.
