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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. Induction of orofacial neuropathic pain and evaluation of mechanical hypersensitivity
NOTE: Following baseline measurements, rats underwent chronic constriction injury of the infraorbital nerves (CCI-ION) surgery, which involved bilateral ligation of the infraorbital nerves (ION), as previously described. Control rats had sham surgery. No pre- or post-operative analgesia was used in the procedure as it can change the time course of the neuropathy. CAUTION: Waste isoflurane must be scavenged through charcoal canisters. Scalpel blades and needles must be disposed of in biohazard waste.
<ol>
	<li>Anesthetize the rat in the induction chamber with a mixture of O2 (1 L/min) and 4% isoflurane and maintain the anesthetic state with a specialized nose cone for the duration of the surgery.</li>
	<li>Place the anesthetized rat on a surgical workbench and restrain it. Maintain the body temperature at 37 &#176;C using a heating pad. Apply ophthalmic ointment to the eyes to prevent them from drying out. Check the anesthetic depth by pinching the toe and start the procedure when the toe withdrawal reflex is no longer observed.</li>
	<li>Perform the surgical procedure under a stereo microscope. Open the mouth using retractors and retract the lip using a small clip.</li>
	<li>Make a small incision between the dorsal gum and lip using a scalpel blade (#15). Gently cut away soft tissue using the tip of the scalpel blade to reveal a branch of the ION.</li>
	<li>Place two chromic gut (#5-0) ligatures around the ION using a blunt, bent syringe needle.</li>
	<li>Close the wound using tissue adhesive.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chromic gut suture size 5-0</td>
			<td>Ethicon</td>
			<td>687-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-8440</td>
			<td>Pivetal</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra</td>
			<td></td>
			<td>Puralube Vet Ointment, petrolatum ophthalmic ointment</td>
		</tr>
		<tr>
			<td>Scalpel blade #15</td>
			<td>FST</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia system</td>
			<td>VetFlo</td>
			<td>VetFlo-1205S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats, female</td>
			<td>Charles River Laboratories, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo boom microscope</td>
			<td>Omano</td>
			<td>OM2300S-GX4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Water circulating heating pad and pump</td>
			<td>Gaymar</td>
			<td>Model TP-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing orofacial neuropathic pain in a rat model via infraorbital nerve ligations

Source: Donertas-Ayaz, B. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64221">Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay.</a> J. Vis. Exp. (2022)
This video demonstrates the induction of orofacial neuropathic pain in a rat model by ligating the infraorbital nerve. This results in nerve damage and inflammation, causing abnormal pain signaling and mimicking chronic pain conditions, which can be used to study the orofacial pain assay.

Inducing Orofacial Neuropathic Pain in a Rat Model via Infraorbital Nerve Ligations

Source: Donertas-Ayaz, B. et al., Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay. J. Vis. ...

Begin with an anesthetized rat restrained on a surgical bench.
Apply an ointment to the eyes to prevent dryness.
Use retractors to open the mouth and retract the lip.
Next, make an incision between the dorsal gum and lip, and remove the soft tissue to expose a branch of the infraorbital nerve.
This nerve transmits the orofacial signals from the face and mouth to the brain.
Place two ligatures around the infraorbital nerve to constrict it, which results in nerve injury and axonal damage.
The injured nerve triggers the release of pro-inflammatory molecules by the immune cells. These molecules attract more immune cells and lead to an exaggerated immune response.
This increases the excitability of the damaged nerve and generates intense signals that travel to the brain. These intense signals are interpreted as neuropathic pain.
Apply an adhesive to close the wound. The rat is now ready for the orofacial pain assay.

inducing-orofacial-neuropathic-pain-rat-model-via-infraorbital-nerve

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

25 vues. Source: Donertas-Ayaz, B. et al., Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay. J. Vis. Exp. (2022) This video demonstrates the induction of orofacial neuropathic pain in a rat model by ligating the infraorbital nerve. This results in nerve damage and inflammation, causing abnormal pain signaling and mimicking chronic pain conditions, which can be used to study the orofacial pain assay. 

Vidéo: Inducing Orofacial Neuropathic Pain in a Rat Model via Infraorbital Nerve Ligations - Concept

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Évaluation de l’hypersensibilité mécanique induite par une lésion nerveuse chez le rat à l’aide d’un test de douleur opérante orofaciale

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

Recherche

JoVE Journal

Sciences du comportement

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Constriction chronique blessures de orbitaire du nerf de la Rat (ion-CCI) d&#39;étudier trijumeau douleur neuropathique

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

Médecine

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Test mécanique d’évitement des conflits pour mesurer le comportement de la douleur chez la souris

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Inducing Orofacial Neuropathic Pain in a Rat Model via Infraorbital Nerve Ligations

Transcription

Inducing Orofacial Neuropathic Pain in a Rat Model via Infraorbital Nerve Ligations

Évaluation de l’hypersensibilité mécanique induite par une lésion nerveuse chez le rat à l’aide d’un test de douleur opérante orofaciale

Constriction chronique blessures de orbitaire du nerf de la Rat (ion-CCI) d'étudier trijumeau douleur neuropathique

Test mécanique d’évitement des conflits pour mesurer le comportement de la douleur chez la souris