Trigeminal neuralgia is often a therapeutic challenge in clinical practice because of the current lack of understanding of the pathophysiological mechanisms involved. Most clinical studies posit that chronic facial neuropathic pain is caused by compression of the trigeminal nerve root.1–4 In some cases, however, trigeminal neuralgia was observed secondary to irritation of trigeminal terminals by oral and dental disease.5,6 Studies elucidating the etiology and pathophysiology of trigeminal neuralgia have been reported based on a variety of animal models. In other models, various lesions of the trigeminal nerve were produced: by infraorbital nerve (ION) loose ligation,7 application of complete Freund adjuvant (CFA) to the trigeminal root,8 removal of tooth pulp from cats,9 and implanting the trigeminal root with chromic suture.10 These models have increased our understanding of the mechanisms of trigeminal neuralgia; however, one animal model cannot mimic all of the sensory, motor, and molecular changes seen.11,12 Therefore, a major problem in further exploring the pathophysiological mechanisms of trigeminal neuralgia has been the lack of a reproducible animal model that demonstrates the observed sensory, motor, and molecular changes.
Previous animal models of trigeminal neuralgia have demonstrated various shortcomings attributed to multiple factors. One laboratory model of trigeminal neuralgia, induced by chronic constriction of the ION, results in neuropathic pain syndrome within the territory of the injured nerve.13 The difficulty with this model is that the degree of damage relies on the accurate manipulation of operators to avoid complete denervation. The surgical procedure is complicated, relatively difficult to perform, and may not be reproducible across various investigations. Another model described by Gobel and Binck9 uses cat tooth pulp removal, leading to degenerative changes in the primary trigeminal nucleus. In contrast to methods used to create trigeminal neuropathic pain by compression or amputation of afferent nerves, an alternative approach has been the use of chemical irritation of the trigeminal nucleus caudalis.8,14 However, inflammatory agents used in previous animal models may not be applicable to human subject research.
In several reports, cobra venom was studied for its effects on the nervous system. Cobra venom contains multiple active substances, such as phospholipase A2 (PLA2), cardiotoxin (CTX), and neurotoxin (cobrotoxin). Zhu et al.15 reported that application of cobra venom to the sciatic nerve led to demyelination of myelinated fibers. Moreover, cobra venom produced inhibition of axonal conduction whereas C-fiber reflex remained unchanged.15,16 Similarly, electrophysiological recordings revealed that ectopic discharges could be elicited from the dorsal roots with the accompanying loss of myelin after epidural administration of PLA2.17 These studies suggest that cobra venom may be appropriately used for studying mechanisms of persistent neuropathic pain.
In this study, we developed a model for producing trigeminal neuralgia in the rat. Cobra venom was applied to the rats' ION trunk to produce some characteristics of trigeminal neuralgia. The procedure is technically easy to perform. It is hoped that this model may provide further insights into the mechanisms of trigeminal neuralgia in humans.
The investigations were approved by the Institutional Animal Care and Use Committee of Tsinghua University (Beijing, China). Also, the entire experimental procedure on conscious animals was consistent with the Guidelines of the International Association for the Study of Pain.18 Male Sprague-Dawley albino rats (weighing 180–220 g) provided by the Laboratory Animal Center of the Academy of Military Medical Sciences were used in this study. The animals were housed with food and water available ad libitum, and kept under a light/dark cycle (12 hours/12 hours) under controlled temperature conditions (23°C–25°C). Thirty rats were randomly divided into 2 groups: (1) cobra venom group, and (2) saline control group. In 15 rats, the cobra venom solution was injected unilaterally into the sheath of the ION (n = 8, behavioral test; n = 7, Evans blue permeability). Rats in the control group (n = 8, behavioral test; n = 7, Evans blue permeability) received 0.9% sterile saline injection.
Cobra venom consisted of lyophilized whole venom of cobra venom (Formosan cobra; Sigma, St. Louis, MO) dissolved in 0.9% sterile saline. A volume of 4 μL saline containing 0.4 mg lyophilized whole venom was used during the entire experiment.
Rats were anesthetized with sodium pentobarbital (40 mg/kg intraperitoneally). The head of each rat was fixed and mounted on an operating table in a prone position. All of the procedures were performed under direct visualization. The incision was made approximately 1 cm in length radically along the superciliary arch superior to the skin margin, exposing the fossa orbitalis and nasal bone. The content of the orbit was then deflected with a glass rod. The ION was freely dissected at its rostral extent in the orbital cavity and raised gently by a glass nerve dissector, while the saline or cobra venom was injected into the nerve sheath of the ION. To avoid leakage of the cobra venom, several slivers were set around the injection site. The incision was closed using 5-0 absorbable sutures.
Examination of Mechanical Allodynia
In the behavioral test, rats were habituated to the test procedure 3 days before operation, and tested for mechanical allodynia 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, and 90 days after the operation. Animals were placed individually in transparent plastic cages (27 × 25 × 20 cm) and habituated for 15 minutes. Mechanical stimuli were applied using a logarithmic series of 8 calibrated Semmes-Weinstein monofilaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g) (von Frey hairs; Stoelting, Chicago, IL). When the rat stopped exploratory behavior and ceased major grooming, the stimuli were applied within the ION territory, near the center of the vibrissal pad on hairy skin surrounding the mystacial vibrissae.19–21 The experimenter reached into the cage very slowly with a von Frey hair. A von Frey hair was held for approximately 5 seconds and presented at intervals of several seconds based on the elimination of the behavioral responses to previous stimuli. A clear withdrawal response was recognized as follows: (1) rat avoids further contact with the monofilament, (2) rat turns head rapidly away scratching the stimulated area, or (3) rat actively attacks the stimulating object. The mechanical threshold of withdrawal response was measured according to the up-down method and thus the 50% threshold was computed using the formula described by Dixon.22 The changes of mechanical response were expressed as the difference between ipsilateral and contralateral sides.
In this study, Evans blue dye was used as a marker for determining vascular permeability. Evans blue dye (Sigma) was prepared by dissolution into sterile 0.9% saline (50 mg/mL), and filtering through a 5-μm filter (Millipore, Bedford, MA). Rats were injected with 5% Evans blue dye (50 mg/kg) via the femoral vein for 2 minutes. After Evans blue dye infusion, the rats turned visibly blue. The procedure for exposure of the ION was performed as described above. Subsequently, a volume of 4 μL saline or cobra venom (0.1 mg/μL) was injected into the sheath of the ION. At 30 minutes after the operation, rats were killed by an overdose injection of sodium pentobarbital (100 mg/kg intraperitoneally) and lavaged transcardially with 800 mL normal saline. The facial skin territory of the ION was removed, and the Evans blue dye was extracted by immersion in quantitative N,N-dimethylformamide at 55°C for 24 hours. The extract from individual skin samples was centrifuged and measured with a spectrophotometer (SmartSpec™ 3000; Bio-Rad, Inc., Hercules, CA) at 620 nm, the absorption maximum for Evans blue dye in formamide.23 The concentration of dye in the skin was calculated from a standard curve of Evans blue in N,N-dimethylformamide. Results are expressed in micrograms of Evans blue dye per gram of skin sample.
Results are presented as the mean ± SEM. Changes in mechanical response thresholds throughout the study period within each group were analyzed using the Wilcoxon signed rank test. The mean mechanical thresholds before and after the operation between groups were analyzed nonparametrically by using the individual Mann-Whitney U test. For statistical analyses of parametric data, the Student t test was used. Statistical significance was determined to be P < 0.05.
As shown in Figure 1, administration of cobra venom produced an obvious decreased response threshold throughout the 90-day postoperative period. Compared with the preoperative value, the cobra venom group demonstrated a profound and prolonged decrease in the mechanical threshold of the ipsilateral ION territory from day 3 after surgery (P = 0.001, Mann-Whitney U test; Fig. 1A). The changes consisted of an initial decrease (on postoperative day 3 to 60) and a subsequent gradual reversal (around postoperative day 70). At the contralateral side, there was also a dramatic decrease in threshold to mechanical stimulation of the territory of the ION from day 3 to 25 postoperation (Mann-Whitney U test; Fig. 1B). During the postoperative period, the control group exhibited no significant changes on either the ipsilateral or the contralateral side. Wilcoxon signed rank test revealed the noticeable differences in threshold between the 2 groups at the test time point of 3 to 60 days on the ipsilateral side. Thereafter, the threshold returned gradually to the preoperative values and became normal 90 days after surgery.
Evans Blue Permeability
Figure 2 shows the extravasation of Evans blue dye in the skin visualized after injection of cobra venom into the ION trunk (Fig. 2B). In contrast, there was absolutely no blue staining of the skin in the control group rats (Fig. 2A). The vascular Evans blue dye leakage in the ION territory was 0.61 ± 0.09 and 0.25 ± 0.03 μg Evans blue × g skin sample dry weight−1 from the cobra venom group and saline group, respectively (Fig. 2C; P = 0.003, n = 7 rats for each group). When comparing the effects of cobra venom on vascular leakage, it was found that the magnitude of extravasated Evans blue dye in the ION territory skin in the rats treated with cobra venom was significantly higher than that of control rats.
Body Weight Change
On the day of surgery, rats were weighed and no statistically significant difference was observed between the 2 groups (control: 191.9 ± 2.0 g; cobra venom: 190.4 ± 2.9 g; n = 8 for each group). However, from postoperative day 3, rats treated with cobra venom (2.5 ± 0.1 g/d) had a lower mean weight gain than control rats (3.0 ± 0.1 g/d) (unpaired t test, P < 0.05). At the end of the observation period (on postoperative day 90), the cobra venom group rats (418.6 ± 8.34 g) had gained less weight compared with the control group (466.1 ± 9.8 g) (unpaired t test, P < 0.05).
Our study demonstrated that after administration of cobra venom into the ION trunk, rats exhibited mechanical allodynia within the distribution of the ION. Furthermore, this hypersensitivity was stable and lasted approximately 60 days on the ipsilateral side, whereas on the contralateral side hypersensitivity was less pronounced over 30 days. There were no changes in mechanical thresholds on either side of the saline-treated rats. In addition, cobra venom–treated rats gained less weight than control rats.
The evoked neuropathic pain behavior investigated in this test is consistent with other animal models of trigeminal neuralgia. It has been observed that rats with a chronic constriction injury to the ION exhibit increased responsiveness to stimulation of this area and spontaneous behavioral abnormalities.7,13,24 Applying CFA to the orbital portion of the ION25 or temporomandibular joint26 also induces significant increased sensitivity to mechanical stimuli in the facial skin. As reported previously, mechanical allodynia that presents in various animal models is representative of the symptoms in patients who have trigeminal neuralgia.27,28 The results observed in our study confirm that this experimental model results in typically seen features of trigeminal neuropathic pain.
The mechanisms underlying the allodynic behavior after administration of cobra venom are poorly understood. It is hypothesized that one mechanism of trigeminal neuralgia may involve the trigeminal dorsal root reflex (DRR) and ectopic action potentials at sites of demyelination.29 This assumption may also explain the hyperalgesia and allodynia observed in neuropathic pain models, such as chronic constriction injury,30 spinal nerve ligation,31 and diabetes disease.32 However, there are some concerns that are not explained. Wallace et al.33 found that demyelination of afferent A fibers was not necessary for development of pain behavior. In addition, ectopic discharge and mechanosensitivity were induced in myelinated axons with no axonal damage in CFA-induced inflammation rats.34 This observation implies that the morphological change of afferent fibers may be only partially responsible for neuropathic pain. This discrepancy might be attributable to the action of different mechanisms operative in diverse animal models.
Cobra venom is a complex molecular compound that may produce several biological and physiological effects. The effects of the main ingredients in cobra venom are CTX, which induces conduction blockade of the nerves, and PLA2, which hydrolyzes phospholipids in the axonal membrane with the aid of CTX. CTX highly binds with the protein layer surrounding axons, thus resulting in changes of the membrane structure.16 These potent components may contribute to the observed disturbances of nerve function.
Using the cobra venom model (cobra venom application to the rat ION), we observed both mechanical hypersensitivity and an inflammatory response within the distribution of the ION. In the Evans blue dye test, a significant increase of blue dye extravasation was present after the application of cobra venom. This suggests that an inflammatory response is evoked by this method. Several studies found that inflammatory responses frequently accompany the development of neuropathic pain.35,36 Based on preclinical observations, one theory posited is that activation of trigeminal sensory fibers leads to painful neurogenic inflammation, which may involve the pain accompanying migraine attacks.37 Previous studies revealed that the release of vasoactive peptides from the terminal endings of afferent A-δ and C fibers by antidromic action potentials or DRRs enhances the inflammatory response (i.e., neurogenic inflammation or axon reflexes).38–42 Moreover, it has been suggested that blockade of the myelinated afferent fibers resulted in an increased discharge of C fibers.43–45 These findings suggest the possibility that the demyelination of A fibers may trigger the C-fiber–mediated DRRs, thus leading to inflammatory action, which subsequently enhances the pain sensation of allodynia. This may be an important factor in the etiology of the development of mechanical allodynia in trigeminal neuralgia.
The present study demonstrates that this method is easily performed and satisfies many of the requirements for studying pain mechanisms in animals with trigeminal neuralgia. Cobra venom is a natural substance that does not produce chemical injury. Additionally, several action components isolated from cobra venom have been used in the treatment of human diseases. Therefore, the use of this test in studying the treatment of trigeminal neuralgia for humans is both practical and reproducible.
In conclusion, this is the first report of the application of cobra venom to establish an animal model of trigeminal neuralgia. The resultant prolonged mechanical allodynia and vasodilation and similarities to clinical syndromes indicate that this model may provide a useful tool to gain insight into the mechanisms and treatment of trigeminal neuralgia.
Name: Jian-Xiong An, MD.
Contribution: Study design, data analysis, and manuscript preparation.
Name: Ying He, MD.
Contribution: Conduct of study, data analysis, and manuscript preparation.
Name: Xiao-Yan Qian, CRNA, BSN.
Contribution: Conduct of study, data analysis, and manuscript preparation.
Name: Jian-Ping Wu, MD.
Contribution: Conduct of study.
Name: Yi-Kuan Xie, BS.
Contribution: Study design, conduct of study.
Name: Qu-Lian Guo, MD, PhD.
Contribution: Study design.
Name: John P. Williams, MD.
Contribution: Study design.
Name: Doris K. Cope, MD.
Contribution: Manuscript preparation.
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