Capsaicin Combined with Local Anesthetics Preferentially Prolongs Sensory/Nociceptive Block in Rat Sciatic Nerve

Capsaicin, amitriptyline hydrochloride, bupivacaine hydrochloride, and lidocaine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). N -Methyl amitriptyline was custom synthesized by Sigma Chemical Co.; the purity was greater than 99% by high-performance liquid chromatography, and the molecular weight was 372.3. Capsaicin was freshly prepared with a solvent of 10% ethanol, 10% Tween 80, and 80% normal saline (pH of the final solution was 6.6). All other drugs were freshly dissolved in 0.9% NaCl (pH ranged from 5.0 to 6.0). The pH was not adjusted because it is probably buffered quickly by the pH of the tissue fluid (7.4).

The animal experimental protocol was approved by the Standing Committee on Animals of Harvard Medical School, Boston, Massachusetts. Male Sprague-Dawley rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and were kept in animal housing facilities with controlled relative humidity (20–30%), at room temperature (24°C), in a 12-h (6:00 am to 6:00 pm) light–dark cycle. Rats were handled before the procedures to familiarize them with the experimental environment and to minimize stress-induced analgesia.15At the time of injection, animals weighed 250–300 g.

The rats were assigned to treatments via  block randomization with a block size of 8. All rats were anesthetized by inhalation of 1–2% of sevoflurane (Abbott Laboratories, North Chicago, IL) until no withdrawal to pinch of the leg occurred (by forceps). After induction of inhalation anesthesia, the drug in a volume of 200 μl was injected at the sciatic notch of the left hind limb with a 27-gauge needle connected to a tuberculin syringe: (1) N -methyl amitriptyline at 0.125%/3.4 mm, (2) amitriptyline at 0.125%/4.0 mm, (3) bupivacaine at 0.25%/7.3 mm, and (4) lidocaine at 2%/73.9 mm. Because sensory/motor separation by LAs is a partially concentration dependent, we obtained dose–response studies for (5) N -methyl amitriptyline and (6) amitriptyline. All drugs were given alone as well as coadministered with capsaicin at 0.05%/1.6 mm. Coadministration of capsaicin was performed either 10 min after the first drug or simultaneously (mixed). The vehicle control group received LA followed by injection of vehicle 10 min later. Capsaicin was also injected alone, as was normal saline. The volume of drug injected was always 0.2 ml. The experimenter was blinded to the drug used (except to the administration of a second drug 10 min later, which was either capsaicin or vehicle alone).

We evaluated motor function and nociception as described previously.16,17Rats were examined before injection for baseline functions and at 10, 20, 30, 60, and 90 min and 2, 3, 6, 12, 24, and 36 h after drug administration. Motor function was assayed by holding the rat upright with the control hind limb extended so that the distal metatarsus and toes of the target leg supported the animal’s weight; the extensor postural thrust was recorded as the force (in grams) applied by each of the two hind limbs to a digital platform balance (Ohaus Lopro; Fisher Scientific, Florham Park, NJ). The reduction in this force, representing reduced extensor muscle contraction caused by motor block, was calculated as a percentage of the control force (preinjection control value 145–165 g). The percent reduction in force was assigned a “range” score: 0 = no block (or baseline); 1 = minimal block, force between the preinjection control value of 100% and 50%; 2 = moderate block, force between 50% of the preinjection control value and 20 g (approximately 20 g represented the approximate weight of the flaccid limb); 3 = complete block, force 20 g or less.

Nociception was evaluated by the nocifensive withdrawal reflex and vocalization to pinch of a skin fold over the lateral metatarsus (cutaneous pain) with a serrated forceps; the force and duration of this pinch was held as constant as possible. The extent of the nocifensive withdrawal reflex and vocalization were combined on a scale of 0–3 for each examination. Grading was as follows: 3 = complete block, no nocifensive reaction or vocalization; 2 = moderate block, vocalization accompanied by slow withdrawal and flexion of the leg; 1 = minimal block, brisk flexion of the leg, with some sideways movement of the body or other escape response and loud vocalization; 0 = baseline with no block and all nocifensive responses listed above.

We restricted our testing of nociception to superficial nociceptive block, i.e. , pinching of a skin fold at the lateral area of the dorsum of the paw, because our pilot studies using pinching of the fifth toe revealed nonreproducible results (data not shown), perhaps because the presumed preferential C-fiber block of various drug combinations does not block large motor (proprioceptive) fibers, which would allow the rat to sense the pressure of the forceps when the entire fifth toe is moved and pinched. However, firm pinch with a serrated forceps to an entire skin fold at the lateral aspect of the dorsum elicited a robust (anti)nociceptive response.

For both nociceptive and motor assessment, the examination was repeated three times at each time point and reported as an average of the three examinations.

Because of the ordinal categorical nature of the block scores, an overall test for drug effect was obtained via  generalized estimating equations for longitudinal ordinal data.18A cumulative logistic ordinal model was fit with a linear and quadratic trend in time and time-by-group interaction. The group effect and group and time interaction effect were tested using contrast coefficients in generalized estimating equations analysis. The overall P  value was calculated via  PROC GENMOD (SAS 9.1; Cary, NC). To have an overall 5% type I error rate for each drug mode, a P  value of less than 0.0167 was considered statistically significant (0.05/3 = 0.0167, because there are 3 sets of comparisons among each drug mode).

After sciatic nerve block, all rats in the treatment groups (n = 8/group) showed a functional loss of nociceptive and motor function of different degrees and durations that were completely reversed over time.

Overall, N -methyl amitriptyline (fig. 1), amitriptyline (fig. 2), bupivacaine (fig. 3), and lidocaine (fig. 4), with injection of capsaicin 10 min later, produced a predominantly nociceptive-specific blockade in this rat sciatic nerve block model. In contrast, simultaneous application of the LAs with capsaicin did not produce a significant differential block, with the exception of N -methyl amitriptyline (tables 1 and 2).

For the 0.125% N -methyl amitriptyline solution when administered alone, the duration of motor and nociceptive blockade was relatively brief (< 1 h) and incomplete. When 0.05% capsaicin was added, either 10 min after the injection of N -methyl amitriptyline or injected simultaneously, a prolonged sensory-selective block resulted (fig. 1).

The duration of the motor block for 0.125% amitriptyline alone was similar to the duration of the nociceptive block, but addition of capsaicin produced a large differential block and significantly decreased the motor blockade when given simultaneously (fig. 2).

Bupivacaine at a concentration of 0.25% was almost indistinguishable from 0.125% amitriptyline, when given alone or in combination with capsaicin. Injection of bupivacaine followed by capsaicin or vehicle 10 min later significantly increased the nociceptive blockade over motor blockade (fig. 3).

Lidocaine at 2% showed a complete but relatively short-lasting motor and nociceptive block when given alone, and the smallest differential block among all drug combinations when the lidocaine injection was followed by capsaicin (fig. 4).

N -Methyl amitriptyline and amitriptyline at concentrations of 0.0625, 0.125, and 0.25% produced dose-dependent and predominantly nociceptive block when the injection was followed by capsaicin (figs. 5 and 6, respectively). With amitriptyline at a concentration of 0.25%, the motor block decreased when followed by capsaicin (fig. 6A).

Injection of capsaicin, normal saline, or vehicle only (solvent of 10% ethanol, 10% Tween 80, and 80% normal saline) caused no detectable block. In addition, injection of the vehicle 10 min after the respective LA produced an immediate and short-lived (2–3 min) intensification of both motor and nociceptive block, with overall no significant differential block (figs. 1–4and table 1).

Intragroup comparison demonstrated significant differences among the different dosing groups (tables 1 and 2). Moreover, the complete recovery time and amount of block data summarized in tables 1 and 2, respectively, show that more hydrophobic drugs such as amitriptyline and bupivacaine (log P  value/octanol buffer coefficient of 4.9 and 3.4, respectively) displayed significantly more differential block than the hydrophilic lidocaine (log P  value of 2.3).

We show that, in addition to the relatively impermeant permanently charged LA QX-314, permanently charged permeant LAs (N -methyl amitriptyline) also produce a pronounced differential rat sciatic nerve blockade when coinjected with or followed by an injection of capsaicin. In addition, tertiary amine LAs also provide enhanced and longer-lasting differential block when followed by capsaicin, and more hydrophobic drugs elicit a larger differential block. Overall, this finding should add to the candidate drug pool available for further preclinical development of sensory-selective LAs.

Several potential mechanisms could explain our findings. The tissue around the sciatic nerve probably has a physiologic pH of 7.4, and therefore the LAs injected around the nerve will produce one of two forms, depending on its pK^a: protonated or neutral. The protonated form is relatively lipid insoluble and therefore cannot penetrate membranes as readily as the neutral form can. The neutral form will penetrate the membrane and, once inside the cell, convert to the protonated form, which blocks sodium channels by binding to the LA receptor located within the inner cavity.19For some LAs, the neutral form may itself also be able to block the channel but is present at lower concentrations. The addition of capsaicin may allow the protonated form to enter the pain fibers selectively through the pore of TRPV1 channels and increase the efficacy and duration of the nociceptive block.

Furthermore, capsaicin-induced activation may lead to the opening of other large pores, such as pannexins,20providing an additional pathway for the protonated form to enter selectively into nociceptors.

Capsaicin-induced depolarization leads to activation and subsequent inactivation of sodium channels, which will “sensitize” them to the effect of LAs by virtue of their higher affinity to inactivated sodium channels.21For example, in vitro , the affinity of amitriptyline, bupivacaine, and lidocaine is approximately 44, 19, and 20 times higher, respectively, for the inactivated state than for the resting state.22,23 

After activation of TRPV1 channels, the cytoplasm of C fibers becomes more acidic24and therefore would increase the charged form of LAs within the cell, which is generally more potent than its neutral counterpart25and leaves the cell more slowly.26Another possibility is that calcium entry associated with TRPV1 activation somehow induces more potent action by the intracellular LAs, perhaps because of changes in the phosphorylation state of the sodium channels.

Our results for permeant LAs demonstrate a nociceptor-predominant sciatic nerve block, but not the nociceptor-selective sciatic nerve block found for the nearly membrane-impermeable LA QX-314 when followed by capsaicin.1This result suggests that capsaicin facilitates the entrance of LAs into the nociceptive nerve fibers through TRPV1 channels but does not interfere substantially with traditional transmembrane crossing of LAs into motor fibers. However, we were surprised to find that simultaneous application of capsaicin decreased the absolute duration of motor block for the more hydrophobic drugs amitriptyline and bupivacaine. The injection of capsaicin could at least temporarily slightly decrease the tissue pH, causing more LA molecules to be positively charged and in turn decreasing the number of LA molecules able to enter the motor nerve fibers. Also, the pK^aof lidocaine (7.8) is lower than those of bupivacaine (8.1) and amitriptyline (9.5). Therefore, a significantly higher percentage of lidocaine will be in the uncharged form and therefore available to block motor fibers, in keeping with our results that showed the largest motor block with the drug of lowest pK^a(lidocaine).

It also seems that the vehicle itself may play a minor role in the nerve blockade. Injection of the vehicle (10% ethanol, 10% Tween 80, and 80% normal saline) 10 min after bupivacaine or lidocaine led to an intensification of both motor and nociceptive block (figs. 3 and 4). This finding is consistent with the known nerve blocking properties of ethanol, and with TRPV1 activation by ethanol.27 

Finally, given that sodium channels are not the only targets of LAs, the effects demonstrated here might be partly due to differential actions of the various tertiary and quaternary agents on K⁺channels, Ca²⁺channels, various ligand-gated channels, second messengers, and substance P neurokinin 1 receptors.28,29 

The finding that the addition of capsaicin to QX-3141produces a nociceptor-selective block has sparked renewed interest in using capsaicin (and its congener resiniferatoxin) to promote differential blockade for regional anesthesia. One concern though is that capsaicin causes a severe burning upon injection. However, in our observations, all rats seemed neurobehaviorally normal upon awakening from a short inhalational anesthesia, as indicated by normal grooming, fluid intake, and exploratory behavior, suggesting that the preceding or concomitant use of LAs eliminated this problem. The path to clinical introduction of novel LAs or LA combinations is usually hampered by toxicity. Although no formal toxicity studies have yet been performed, the overall low concentrations of drugs used by Binshtok et al.  1and in the current study encourage cautious optimism, as does the full return to baseline.

Sciatic nerve block with quaternary ammonium and tertiary amine LAs followed by injection of capsaicin provides a predominantly sensory/nociceptor selective block with a duration that greatly exceeds that produced by the LA alone. Therefore, exploitation of the interaction of TRPV1 receptor agonists and several chemically distinct groups of LAs seems to be a promising path toward regional analgesia without motor block. Besides capsaicin, other TRPV1 channels activators and timings of injection in small and large animals will need to be examined to find the optimal concentrations and timing of combinations of LAs and TRPV1 agonists for clinical use.