A primary sensory nerve is the first afferent pathway beginning at the receptor and ending at a synapse, with a secondary sensory nerve to transfer messages to the central nervous system. It is also an efferent pathway (1,2). Its action in the periphery mediates the nociception system and liberates neurotransmitters, such as substance P, neurokinin A, neurokinin B, and calcitonin gene-related peptide (2,3). These neuropeptides act on peripheral target cells, such as mast cells, immune cells, endothelial and vascular smooth muscle cells, producing inflammation, which is characterized by redness, warmth, swelling, and hypersensitivity (4,5). This phenomenon, called “neurogenic inflammation,” is thought to be involved in a number of diseases including migraine (6), arthritis (7), chronic obstructive pulmonary disease, including asthma (8), inflammatory bowel disease (9), and reflex sympathetic dystrophy syndrome (10).
There are several studies demonstrating the antiinflammatory effects of local anesthetics on neurogenic inflammation. An inhibitory effect of intraarticular lidocaine on bradykinin-induced plasma extravasation was demonstrated in a knee-perfusion model (11). The neurogenic inflammatory response in rat skin was decreased by the application of lidocaine (12). Other local anesthetics, such as ropivacaine and lidocaine-prilocaine cream, also have significant antiinflammatory effects (13,14). However, some studies have failed to demonstrate the antiinflammatory effects of local anesthetics on neurogenic inflammation. Lidocaine or bupivacaine injected into a rat temporomandibular joint displayed no inhibition of mustard oil-induced edema (15). Lidocaine also failed to inhibit human nasal mucosa plasma extravasation induced by bradykinin (16). Furthermore, rat knee joint plasma extravasation produced by a platelet-activating factor was not affected by lidocaine (11). Thus, the effects of local anesthetics on neurogenic inflammation are inconsistent. However, there is little information about the direct effects of local anesthetics on the primary sensory nerve. Therefore, in the present study, we investigated this area by using more direct methods.
Rabbit iris sphincter muscle is innervated by the trigeminal nerve, in addition to parasympathetic nerves, which produces tachykininergic and cholinergic contractions, respectively (17,18). Rabbit iris tissue is therefore a useful preparation to analyze sensory nerve-mediated peripheral responses that are involved in inflammation via tachykinins (19,20). In the present study, we investigated the effects of the local anesthetics, cocaine (the ester class), and lidocaine (the amide class) on trigeminal tachykininergic responses in the rabbit iris sphincter muscle and compared them with parasympathetic cholinergic responses.
Twelve irises from six male albino rabbits (Charles River Japan Inc, Japan) weighing 2.0–3.0 kg were used. The experimental protocol was approved by the institutional animal care committee of Fukui University.
After inducing anesthesia with pentobarbital, rabbits were exsanguinated, and the eyes were immediately isolated. A sphincter muscle strip was prepared from one eye, as previously described (17), and then mounted in an organ chamber containing 20 mL of modified Krebs-Henseleit solution (in [mM]: NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2, and glucose 10; pH value of 7.4) bubbled with 95% O2–5% CO2 at 37°C, and the changes in tension were recorded isometrically. Strips were equilibrated for more than 1 h before the start of the experiments. A resting tension of 150 mg was maintained throughout the experiments.
Electrical transmural stimulation (ETS) was applied via a pair of platinum electrodes (15 mm long, 0.6 mm in diameter, and 3 mm distance between the electrodes) through a current booster with an electronic stimulator (Nihon Koden, SEN-3201). Stimulus parameters were square wave pulses of 0.2 ms in duration and 7.5 V in intensity for 10 s. The stimulus frequencies were 3, 10, and 30 Hz, unless otherwise mentioned. Stimulation was applied at 10- to 15-min intervals. To abolish cholinergic and tachykininergic responses, the iris preparations were treated with 1 μM of atropine or 10 μM of [D-Pro2, D-Trp7,9]-substance P (a tachykinin receptor antagonist), respectively.
Cumulative concentration response curves of carbachol or neurokinin A were obtained by increasing concentrations of the drugs as soon as a steady response to the previous administration had been achieved.
The iris preparations were treated with cocaine or lidocaine in the organ chamber for 30 min before recording the responses to ETS, carbachol, or neurokinin A. Responses were expressed as a percentage of the maximum contractions induced by ETS, carbachol, or neurokinin A before treatment with cocaine or lidocaine.
The following drugs were used: atropine sulfate, carbachol hydrochloride, lidocaine hydrochloride, capsaicin (Sigma Chemical Co, St. Louis, MO), [D-Pro2, D-Trp7,9]-substance P, neurokinin A (Peptide Institute, Osaka, Japan), and cocaine (Takeda, Osaka, Japan).
The results are expressed as mean ± sd. To evaluate differences among groups, one-way analysis of variance was used. When significant differences were detected by analysis of variance, the Scheffé F test was applied for post hoc comparisons. Statistical significance was assumed at a P value <0.05. Analyses were performed on a personal computer using Stat View II 4.0 software (Abacus Concepts, Berkeley, CA).
In isolated rabbit iris sphincter muscle, an ETS (10 Hz for 10 s) produced a similar level of contraction for each experiment (298 ± 23 mg). The contraction was composed of the fast twitch contraction as the first component overlapped with the slow second contraction (Fig. 1). The fast contraction was abolished by 1 μM of atropine, and the residual slow component was inhibited by both 10 μM of [D-Pro2, D-Trp7,9]-substance P and pretreatment with 10 μM of capsaicin (data not shown). In the following experiments, each component was selectively inhibited either by atropine or [D-Pro2, D-Trp7,9]-substance P, and the effects of cocaine and lidocaine on both responses were examined. Figure 2 shows concentration-dependent inhibition of cocaine and lidocaine on two components where the preparations were stimulated at 10 Hz. The inhibition at the same concentration of each local anesthetic was more evident in tachykininergic than in cholinergic contractions.
Next, stimulus frequency-dependency in the inhibitory effects was examined. As shown in Figures 3 and 4, cocaine and lidocaine produced greater inhibitions at higher frequencies of ETS, and the inhibition was more evident in tachykininergic than in cholinergic contractions.
No drugs changed the resting tension throughout the experiments.
Carbachol and neurokinin A produced concentration-dependent contractions in rabbit iris sphincter muscle (Figs. 5 and 6). Cocaine and lidocaine at 100 μM did not affect the contractile response to carbachol or neurokinin A. Maximal absolute contraction strength of carbachol (100 μM) and neurokinin A (300 μM) were 455 ± 49 mg and 438 ± 34 mg, respectively.
The rabbit iris sphincter muscle is innervated by parasympathetic oculometer and trigeminal sensory nerves, which produce cholinergic and tachykininergic contractions, respectively (2,17,21). The tachykininergic contractions are mediated by tachykinins, including neurokinin A, released from the trigeminal sensory nerves, and are capsaicin sensitive (18,19). It was confirmed with atropine and [D-Pro2, D-Trp7,9]-substance P that these responses were composed of the fast cholinergic and slow tachykininergic components (Fig. 1). Two local anesthetics, cocaine and lidocaine, attenuated both the contractile responses to ETS (Fig. 2), and the inhibition was frequency-dependent on nerve stimulation (Figs. 3 and 4). Exogenously applied carbachol and neurokinin A contracted sphincter muscle dose-dependently, but the contractile responses were not affected by cocaine and lidocaine (Figs. 5 and 6). Therefore, the attenuation in the responses to ETS by local anesthetics is predominantly caused by the inhibition of presynaptic, but not postsynaptic, processes.
The main site of action of local anesthetics is Na channels in the nerve axon. However, local anesthetics have been reported to have no selectivity among Na channels of different kinds of nerves. Instead, the firing rate of the action potential and the spacing of nodes of Ranvier are considered the main factors determining the efficacy of local anesthetics (22). In the present study, cocaine and lidocaine produced greater attenuation in ETS responses at higher stimulus frequencies (Figs. 3 and 4). Both the trigeminal and oculomotor nerves innervating the iris sphincter muscle are grouped into unmyelinated C fibers. However, the tachykininergic response was attenuated by smaller concentrations of cocaine and lidocaine as compared with those in the cholinergic response. Therefore, there may be alternative mechanisms causing a high sensitivity to local anesthetics in the trigeminal sensory nerve. Because parasympathetic postganglionic fiber seems to be shorter than sensory fiber remaining in the isolated iris sphincter muscle (17), one possible cause of different sensitivities may be the different length of axon exposed to local anesthetics (23). However, further studies are required to explore the possibility.
There is a report that tachykinin antagonists also work as local anesthetics (24). In the present study, [D-Pro2, D-Trp7,9]-substance P inhibited only the tachykininergic response but not the cholinergic response. This result may indicate that the cholinergic response would tolerate local anesthetics well if the tachykinin antagonist worked, in part, as a local anesthetic.
It is interesting to note that the contractile responses to ETS are blocked by much smaller concentrations of local anesthetics than those for topical use in pain management because such small concentrations could have a therapeutic application in the treatment of peripheral neurogenic disorders caused via primary sensory nerves. Small concentrations of lidocaine administered systemically are effective for treating neuropathic pain (25), which is, at least in part, associated with the peripheral activation of primary sensory nerves and subsequent inflammation.
In conclusion, the present study shows that local anesthetics inhibited the peripheral transmission of the trigeminal sensory nerve on presynaptic processes and that the inhibition was stronger than that for the transmission of the parasympathetic nerve.
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