Temporomandibular disorders (TMD) and rheumatoid arthritis (RA) are diagnosed by signs and symptoms, including clicking, crepitus, pain, and limitation of joint movement (1,2). The use of symptoms to diagnose TMD or RA of the temporomandibular joint (TMJ) is ultimately a reflection of limited appreciation of pathophysiological mechanisms underlying these conditions. Not surprisingly, current management approaches are unsatisfactory in terms of diagnostic criteria and long-term clinical outcome (3). The hypothesis that neurogenic inflammation (NI) plays a pivotal role in the development of TMD and RA has been increasingly accepted in recent years (4).
NI is defined as increased vascular permeability and plasma extravasation (PE) elicited by antidromic stimulation of nociceptive nerve fibers (5). This efferent function of afferent nerves has been proposed to be manifested by means of a neurogenic “dorsal root reflex”(6). Orthodromically conducted action potentials elicited from afferent nerve terminals into the central nervous system are thought to result in impulses antidromically, leading to the release of proinflammatory mediators. The sympathetic nervous system has been suggested to play a significant, though as yet undefined, role in modulating NI (7,8). The central nervous system also appears to influence NI development. For example, unilateral arthritis development in hemiplegic patients has been reported in several instances (9–11).
Evidence for NI in the TMJ has focused on identifying putative proinflammatory neuropeptides such as substance P (SP). Comparisons of TMJ aspirates from patients with internal disk derangement and control groups have revealed more intense expression of SP-like immunoreactivity and pain ratings from affected patients (12). SP injection into the TMJ increases articular levels of other proinflammatory peptides, such as neuropeptide Y, neurokinin A, and calcitonin gene-related peptide (13).
Despite recent interest in the study of the relevance of NI in TMD and RA, the aforementioned evidence for proinflammatory neurogenic mechanisms in the TMJ has been circumstantial at best. Direct physiologic examination of the TMJ to characterize the potential role of neurogenic mechanisms in inducing inflammation appears to be the subsequent logical step for investigation.
Mustard oil (MO) (allyl isothiocyanate) is a selective excitant of small unmyelinated nociceptive fibers, which appears to act solely through a neurogenic mechanism (13). Inflammation elicited by MO is not directly through arachidonic acid metabolism, mast cell degranulation, histamine, serotonin, nitric oxide, or calcitonin gene-related peptide receptor mechanisms (14,15). Hu et al. (16) demonstrated that MO can induce an increase in rat jaw or neck muscle electromyographic (EMG) activity in addition to inducing an acute inflammatory reaction characterized by PE. Theoretically, blockade of PE should be possible by preventing depolarization of neural membranes by local anesthesia. The action of local anesthetics, such as lidocaine, is at the neural membrane surface, at the site of specific receptors located on or within sodium channels (17). Receptor binding prevents influx of sodium through these channels and inhibition of neuronal membrane depolarization.
Our purpose for this study was to directly investigate the neurogenic contribution underlying acute TMJ inflammation by evaluating the effect of local anesthetic blockade on the development of MO-induced edema in the rat TMJ area.
All surgeries and procedures were approved by the University of Toronto Animal Care Committee in accordance with the regulations of the Ontario Animal Research Act (Canada). The methods used have been modified by those described previously by Fiorentino et al. (18). Male Sprague-Dawley rats weighing 275 to 450 g were housed in pairs in constant humidity, temperature of 20°C, and light and dark cycles of 12-h duration. Rats were permitted access to food and water ad libitum and allowed 1 wk to become acclimated to their surroundings before experimental procedures. Rats were weighed immediately before anesthesia. Anesthesia was induced by intraperitoneal injection of α-chloralose (50 mg/kg) and urethane (1000 mg/kg) solution. Rectal temperature was maintained at 37.2° ± 0.5°C and heart rates between 350 and 400 bpm. A tracheal cannula to facilitate maintenance of a patent airway during the experiment was inserted into the proximal airway through an incision made just below the third tracheal cartilage and sutured in place with 2-0 silk. A surgical approach was initiated to place an IV cannula in the left femoral vein. Skulls were stabilized by a stereotaxic appliance and a dental plaster mold which engaged the left side of the head and neck for additional stabilization. A double-barrel cannula constructed from two 27-gauge dental needles connected to PE50 polyethylene tubing attached to two 25-μL glass Hamilton syringes was inserted into the right peritemporomandibular area. An ultrafine needle was bent 90° 3 mm from the tip and the proximal end attached to the skin overlying the right TMJ area with a drop of cyanoacrylate adhesive. The distal end of this needle was allowed to lie passively on a paper support track. This served as a physical marker of lateral tissue expansion. A second needle was fixed to a micromanipulator and the tip lined up the marker with the aid of a dissecting microscope. After swelling of the periarticular tissues, the marker fixed to the rat tissue became displaced, allowing quantification of edema. An injection of 10 μL of saline or local anesthetic (5% lidocaine or 0.5% bupivacaine) at time0 into the peritemporomandibular area was followed by MO (1% to 60%) at time6. Readings were taken every 2 min from time0 to time60 and then every 5 min to time150 (Fig. 1a). Each experimental group was composed of eight rats.
Blockade of conduction in TMJ nociceptors was confirmed with the use of an EMG model. Single rats were used in EMG experiments because only all or none EMG responses were sought. Male Sprague-Dawley rats similarly housed, fed, and acclimated as described for the tissue expansion model were anesthetized with a mixture of 1.5% to 2.0% halothane and N2O/O2 (2:1). Skulls were similarly fixed on a stereotaxic device and placement of TMJ, tracheal, and femoral cannula were initiated as described previously. EMG electrodes were placed in anterior digastric and masseter muscles. Bilateral EMG activity was amplified (gain ×500, bandwidth 30–1000 Hz), displayed on an oscilloscope, and processed online with a data acquisition and processing system sampling at 2000 Hz (1401 plus®, and Spike2®, both by CED, Cambridge, UK). All EMG activities were normalized relative to baseline values, and areas under the curve were calculated. Bilateral EMG activities were recorded in both anterior digastric and masseter muscles continuously before, throughout, and after catheter placement.
Increased reflex jaw muscle activity at the time of catheter insertion was considered confirmation that it had entered the joint properly. In addition, the left or right side was evaluated for strength of signal. Pilot work had demonstrated that no reflex increases in EMG activity above baseline were observed if the catheter failed to pass through the joint capsule.
To evaluate the duration of lidocaine, 20 μL of 40% MO was injected at 30, 20, and 10 min (in separate trials) after injection of 10 μL of 5% lidocaine (Fig. 1b). These will subsequently be referred to in Results as t30, t20, and t10, respectively. To evaluate the effect of bupivacaine, 20 μL of 40% MO was injected at 60 and 30 min (in separate trials) after injection of 10 μL of 5% bupivacaine (Fig. 1b). These will subsequently be referred to in Results as t60 and t30, respectively. The presence of increased EMG activity in the anterior digastric and/or masseter muscle above baseline after injection was considered to be an indication of failure of blockade. The relative magnitude of EMG activity was not considered. Conversely, the absence of increased EMG activity in the anterior digastric and/or masseter jaw muscle after injection of MO was considered to be an indication of complete blockade.
At the conclusion of the experiments, Evans blue (EB) dye (10 mg/mL, 20 mg/kg) was injected IV through the femoral cannula 20 min before euthanasia by using T61 (Hoechst, Regina, Saskatchewan, Can-ada). Transcardial perfusion was then performed with 300 mL of 0.9% saline. Postmortem dissection and examination for staining of the disk and capsule were considered as indication of correct catheter placement.
Statistical analysis was performed with the aid of SPSS software (SPSS Inc., Chicago, IL). The determination of significant differences among mean expansion distances from time−15 to time150 were accomplished with a general linear model repeated measures analysis of variance (ANOVA) with Bonferroni correction. A P value <0.05 was used to determine statistical significance.
Postmortem dissection confirmed correct catheter placement for all rats in which the data were used. Data from six rats were not included because EB dye indicated misplacement of joint catheters. All groups in the tissue expansion experiments were composed of eight rats. Before the injection of saline at time0 (t0) until time20 (t20), trauma from catheter insertion produced an equivalent mean baseline expansion in all groups (ANOVA, P > 0.05). Tissue expansion after 150 min was not statistically significantly different among mineral oil, 1% MO, and 2% MO (Groups 1, 2, and 3) (ANOVA, P > 0.05). Mineral oil (Group 1) differed significantly from 20%, 40%, and 60% MO at t150 (Groups 4, 5, and 6) (ANOVA, P < 0.05). A dose/response relationship followed increasing concentrations of MO and periarticular edema for all groups (Fig. 2).
At t150, tissue expansion for lidocaine pretreated rats (Fig. 3, Groups 7 and 8) did not differ significantly from saline controls. (Fig. 3, Groups 2 and 5) (ANOVA, P < 0.05). Similarly, bupivacaine pretreated rats (Group 9) did not differ significantly from saline controls (Fig. 4, Group 5; ANOVA, P > 0.05).
No significant increases in EMG activity were observed during all baseline recording between 0 and 10 min in either masseter or digastric muscles. All EMG experiments were completed with one rat in each group because only all or none response were sought. No significant increases in EMG activity were evoked with local lidocaine injection at t0. No increase in EMG activity in either digastric or masseter muscles was evoked with 40% MO injection 10 or 20 min after lidocaine (Fig. 5a). An acute increase in activity was evident at t30, demonstrating that the blockade was present at 20 min but incomplete at 30 min.
EMG activity evoked by MO injections after bupivacaine are shown in Figure 5b. No activity was evident after injection of 40% MO 30 min after bupivacaine. An acute increase in activity was evident after injection of 40% MO at t60. Complete conduction blockade by bupivacaine was at least 30 min in duration but not longer than 60 min after administration.
This study demonstrates that MO induces dose-dependent edema development in the rat temporomandibular area. Local anesthesia of the temporomandibular area failed to block development of MO-induced edema. In additional experiments, local anesthetic blockade inhibited for up to 60 minutes the reflex EMG activity in masseter and digastric muscles normally induced by MO injection.
Three technical considerations must be considered before interpretation of the aforementioned findings. First, it is necessary to address the possibility that the relatively large concentration of MO (40%) originally used to evoke a maximal degree of inflammation could be acting through predominantly nonneurogenic means. A direct action on immune cells to release inflammatory mediators and/or direct action on vasculature to increase permeability is a possibility, as suggested by Lynn and Shakhanbeh (19). It was deemed appropriate in this study to subsequently use a small concentration of MO (1%, enough to evoke a perceivable edema response) and again apply a local anesthetic block. No difference between control animals receiving saline pretreatment and experimental animals receiving 5% lidocaine pretreatment was observed. The hypothesis that MO acts neurogenically at small but not large concentrations is not supported by these latter observations.
Second, the duration of block may be inadequate to demonstrate a difference between anesthetic and control groups. Subsequent EMG studies demonstrated a complete block between 10 and 20 minutes after lidocaine and at least 30 minutes after bupivacaine administration, yet it failed to inhibit the course of edema development. Furthermore, though complete conduction block is not achieved after these times, it is reasonable to assume blockade is in no way fully dissipated (Fig. 5). Yu et al. (20) demonstrated that the relative magnitude and duration of responses to MO appeared to increase with time after local anesthetic administration, returning to normal levels after more than one hour. Despite this, no difference was observed between controls and rats given local anesthetic pretreatment. The suggestion that duration of complete local anesthetic blockade is too brief to demonstrate a difference is unlikely.
Third, it may be argued that a possible masking effect of PE is present because lidocaine may have inherent vasodilatory properties on the vessels in the TMJ. Lidocaine has a biphasic dose-dependent effect on vasculature (21). In a pilot study, lidocaine injected in the rat TMJ alone did not produce any PE different from saline controls 30 minutes after injection. In the current study, we used a six-minute delay between injection of local anesthetic (t0) and injection of MO (t6). Within this time period, no difference between tissue expansion between bupivacaine or lidocaine and saline controls was seen (P > 0.05, ANOVA). The vasodilating effect of local anesthetic is unlikely to have had a significant impact.
Local anesthesia does not block neurogenic temporomandibular edema induced by MO. In light of current evidence, which indicates that MO acts purely neurogenically (14,17,22–25), this finding appears paradoxical. The results may be interpreted in two alternative ways: MO acts nonneurogenically to produce edema, or MO produces NI independent of axonal depolarization of afferent neurons. Conceivably, the release of mediators of NI by direct action on nociceptive terminals is occurring.
The former suggestion is at odds with indications of selective neurogenic activity of MO in the rat paw skin (24) and mouse ear skin (15). Nonetheless, to consider this possibility is to suggest that the trigeminal system is somehow unique with respect to the ability of afferent nerves to produce PE. This may be a reasonable suggestion because structures of the TMJ are noncutaneous. Other noncutaneous tissues (such as the vas deferens, testes, prostate, and abdominal muscles of the rat) do not exhibit extravascular accumulation of EB dye with antidromic stimulation of lumbar dorsal roots (26).
An alternate explanation for the paradoxical results observed is that NI elicited by MO is through direct release of mediators from nociceptive terminals independent of axonal conduction. Assuming that generation of NI independent of axonal depolarization occurs, an important implication is presented. Traditional theories that hold axonal conduction as fundamental to NI production should be reconsidered. The “dorsal root reflex” theory holds that peripheral (inflammatory) nociceptive inputs sensitize central circuits and in turn produce depolarization of primary afferents antidromically from central terminals. The resulting action potentials release proinflammatory neuropeptides, which serve to exacerbate the primary process that originally provided nociceptive inputs in this way providing for a positive feedback mechanism (6).
MO produces a dose-dependent increase in edema development in the rat TMJ which suggests that there seems to be a neurogenic component to inflammation involving the joint. This inflammation induced by MO cannot be inhibited by local anesthetic blockade (effectively a “functional deafferentation”). Alternatively, this study provides evidence that MO acts nonneurogenically, contrary to century-old dogma.
The implications of such a finding may ultimately be manifested in future therapeutic approaches to inflammatory disease. Antagonism of neurally derived mediators of NI directly, or effecting physical rather than functional deafferentation of affected joints, are examples of two such possibilities. The suggestion that RA and associated TMJ involvement may embody a significant neurogenic component continues to garner attention for future study.
The authors wish to thank Dr. H. Tenenbaum, Dr. B. Cairns, Dr. P. Fiorentino, and Miss B. Cai for their valuable input in production of this manuscript. Technical assistance was provided by Ms. Susan Carter and Mr. Ken Macleod.
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