Lipophilic local anesthetics block axonal nerve conduction and can be effectively used to prevent transmission of nociceptive stimuli without significant motor impairment (1). However, the nonspecificity of the effect and the relatively short duration of action limit the effective application of most local anesthetics in the management of chronic pain. The limitations imposed by the pharmacologic properties of local anesthetics have prompted the investigation of administration methods to prolong duration of analgesia.
Methods that have been applied to prolong the analgesic effect of local anesthetics include liposomal encapsulation, ion complex formation of medium molecular weight molecules, polymer impregnation, and material-based microparticles (2). The latter method has been used using butyl-aminobenzoate (butamben), a highly lipophilic local anesthetic of the ester class, to prepare a suspension that produces a differential nerve block of long duration. Epidurally administered butamben, as a 10% suspension formulated with 2.5% polyethylene glycol 3350 in 0.9% sodium chloride, provided pain relief of up to 34 wk in terminally ill cancer patients (3). Similarly, a 10% suspension of butamben using 0.025% polysorbate 80 as the suspending agent produced prolonged analgesia (8–16 wk) without significant motor or adverse sensory blockade in cancer patients (4). More recently, epidural and peripheral nerve blocks with a 5% butamben suspension, prepared with 2.5% polyethylene glycol 3350 and 0.025% polysorbate 80, provided pain relief of up to 96-wk duration with a concomitant reduction in the need for supplemental opioids (5). A 5% butamben suspension is undergoing Investigational New Drug phase 2 clinical studies.
In the rat paw formalin test, intradermal injections of formalin produce a quantifiable and reproducible biphasic nociceptive response with early flinching of the limb (0–5 min) followed by a quiescent period (10–15 min) and a subsequent tonic or late response (20–40 min) (6,7). Local anesthetics administered before the intradermal injection of formalin nearly ablate the late (tonic phase) response; however, when administered after the early phase they produce a diminished antinociceptive effect (8). The primary goal of this study was to determine the antinociceptive effect and time course of a butamben sciatic nerve block on the late (tonic phase) response to intradermal paw formalin injection, as well as the thermal withdrawal latencies, in the rat. A secondary goal was to examine the histological effect of butamben on the sciatic nerve.
With approval of the institutional animal care and use committee, male Sprague-Dawley rats (200–225 g;n = 116) were randomly divided into 18 groups of 6 and 1 group of 8. The animals were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) and placed in the prone position. Using a 25-gauge short bevel needle connected to a nerve stimulator (Model SD-9; Grass Instrument Co., Quincy, MA) the right sciatic nerve was located by inserting the needle at the palpated sciatic notch. The needle was advanced to the point of maximum response to stimulation at 0.2 V (1.5–2 mA), 2 ms duration and a frequency of 2 Hz. Nine groups of 6 and the 1 group of 8 rats received an injection of 5% butamben (0.3 mL) and 9 received physiologic saline (0.3 mL), administered via the needle used to identify the point of maximal stimulation.
On each of days 1, 2, 5, 10, 21, and 28 after injection one saline group and one butamben group received an intradermal injection of 5 μL of 5% formalin into the dorsum of the paw of the right hind limb. The injection was performed under brief halothane anesthesia (exposure to 4% halothane until loss of righting reflex). Rat behavior, including flinches and licking of the injected paws, were recorded for 1-min epochs at 5-min intervals from 5 min to 60 min by observers blinded to the study group. Before the formalin injections, rats were also evaluated for motor coordination on a rod rotating at 10 rpm (Economex, Columbus Instruments, Columbus, OH). Normal rats are able to maintain their balance on this rod for at least 3 min without falling (9).
In the group of eight animals the withdrawal response to thermal stimulation was evaluated by the plantar stimulation test (10), using the Paw Thermal Stimulator System (University of California, San Diego). Briefly, the animal was placed in a clear container on a heated glass surface (30°C) and allowed to adapt for 30 min before testing. A radiant light source was positioned so that it was focused on the lateral plantar surface of one of the hind paws. After light activation, latencies were recorded as the time until withdrawal. The stimulus intensity was chosen so that the average latency of nociceptive response was 8 ± 0.5 s before sciatic nerve injection. Withdrawal responses were measured in triplicate in both hind paws on the day before sciatic nerve injection and on days 1, 2, 5, 7, 10, 14, 17, 21, and 28 after injection. This group was also evaluated for gross motor dysfunction on a rotating rod before thermal testing as described above.
Histological evaluation of the sciatic nerves was performed in the final 4 groups at 2 time points, 10 days for 2 groups, and 28 days for 2 groups (1 butamben and 1 saline at each time point). The rats were anesthetized with pentobarbital 90 mg/kg and perfused into the aorta with cacodylate-buffered saline (0.9%) 120 mL over 3 min, followed by a dilute aldehyde fixative (1% paraformaldehyde, 1.25% glutaraldehyde, and 0.05% calcium chloride in 0.8 M cacodylate buffer at pH 7.2) for 7 min at the same rate. The nerves on both sides were dissected and trimmed and then divided into proximal, middle, and distal portions, osmicated (2% aqueous). They were stained en bloc with uranyl acetate (1%), dehydrated in graded ethanol, and embedded in Epon-Araldite. Semi-thin sections from each portion of the nerve were cut at 1 micron and stained with methylene blue/azure II for light microscopic examination by a single investigator (JK) who was blinded to the treatment of the groups. Representative specimens were selected for ultrathin sectioning, electron microscopic examination, photography, and analysis.
Data for the number of licks and flinches after intradermal formalin injection as well as withdrawal latencies to thermal challenges were compared using analysis of variance for repeated measures. For the formalin-injected groups, statistical comparisons were made against matched saline groups on the same day postinjection. Responses to thermal challenges were compared against the contralateral noninjected limb. When statistical significance was achieved (P < 0.05), post hoc testing using paired t-tests with Bonferroni’s correction was used to identify significant differences.
Animals that received butamben injections demonstrated decreased formalin-induced nociceptive responses compared with saline control animals on days 2, 5, 10, 21, and 28 (Fig. 1). Butamben-injected animals did not exhibit a reduction in nociceptive responses when compared to saline on day 1 after injection. The magnitude of the butamben effect was greatest on day 10, with a reduction in flinches at all times longer than 20 min after formalin injection. Saline responses were not significantly different on any day.
There were no grossly observable differences in motor function among the groups. Animals had no apparent changes in gait or stance, were able to climb out of open cages, and were able to stay on the rotating rod for 3 min throughout the study. No animals exhibited gnawing or chewing of the foot throughout the study period, had abnormal nail or hair growth, or developed atrophy in the muscles of the injected limb. All groups gained weight at a similar rate throughout the study.
Prolonged withdrawal latencies to thermal challenge to the hind paws were observed for the first 14 days in the butamben-injected limb compared with the contralateral hind paw (Fig. 2). Before butamben injections withdrawal latencies were 7.9 ± 0.3 s and 7.8 ± 0.3 s for the left and right hind paw, respectively. Left side latencies did not increase after right sciatic nerve block throughout the study period.
Histologically, the proximal sciatic nerve at the site of the injection is monofascicular and all but 2 (1 saline 28 days, 1 butamben 10 days) of the 24 nerve specimens had a normal histology comparable to the contralateral noninjected side. Typically distal to the site of injection, where the nerve splits into three fascicles, normal microscopic appearance was evident with both large and small myelinated axons interspersed with clusters of nonmyelinated axons. The typical light microscopic appearance at the distal location is shown at 50× magnification and at 500× magnification with oil immersion (Fig. 3). As typical for a mixed nerve, both large and small myelinated axons are interspersed with clusters of nonmyelinated axons.
The normal pattern was more evident at the ultrastructural level with the thickness of the myelin sheath increasing in proportion to the increase of the axonal diameter. However, this was variable as some small axons with very thin myelin sheaths were also seen in control specimens (Fig. 4). Isolated vacuolation was seen in some myelinated and nonmyelinated axons on both experimental and control sides. In addition, occasional disruptions or redundancies of the myelin sheath, clusters of axonal vesicles and a wide range of cytoskeletal densities were seen in both injected (saline and butamben) and control nerves.
In the two specimens where more extensive anatomical changes were seen, there was evidence of Wallerian degeneration (Figs. 5,6). In these cases, the appearance of many thinly myelinated axons, redundant loops of the myelin sheath, and a redundancy of the basal lamina suggested the presence of a prior focal injury at a more proximal site where the injection was given.
The important findings of this study are the demonstration that sciatic nerve injection of 5% butamben suspension increased the nociceptive threshold to a plantar thermal stimulus for 14 days and reduced the late phase response to the intradermal injection of formalin when administered up to 28 days beforehand. Although formal motor evaluations, other than the ability to maintain balance on a rotating rod for 5 minutes, were not performed, no animal displayed evidence of gross motor impairment or had evidence of obvious anesthesia of the foot. Histologic studies of the sciatic nerve in rats at 10 and 28 days after butamben injection showed minimal effects. Taken together these findings suggest a differential sensory effect of long duration without substantial neuropathology.
Animal models of nociception generally involve a suprathreshold stimulus of short duration and do not allow for development of the neural and biochemical changes that occur with a prolonged stimulus. The rat paw formalin test has two distinct phases, allowing for a more precise examination of analgesic effect. The early phase occurs immediately after injection of formalin in the rat paw and lasts for approximately 5 minutes. During this phase the animals demonstrate nociceptive behaviors such as licking, flinching, and shaking of the injected paw that is considered necessary for development of the late phase response (11,12). The early phase is followed by a quiescent period in which the animals demonstrate minimal pain behaviors. The late phase or second phase follows the quiescent period, occurring approximately 20 minutes after initial injection. This phase is characterized by resumption of the nociceptive (pain) behavior and lasts for an additional 20–40 minutes (13). These results support the theories of central nervous system (dorsal horn) sensitization and plasticity, although continuing afferent input has also been demonstrated to contribute to the late phase response (14).
In this study, the application of the local anesthetic butamben preceded the injection of formalin or thermal challenge by 1 to 28 days. Increased thermal withdrawal latencies were observed the day after butamben injection, but the antinociceptive effect of butamben was not apparent until the administration preceded formalin by two or more days. A prolonged local anesthetic neural blockade before the injection of a noxious chemical in a similar model has been shown to be necessary to suppress late phase nociception (15–17). The latter parallels clinical experience with butamben where the onset of peak analgesia can be delayed for several days after epidural administration or nerve block as measured by reduced pain scores and decreased opioid requirements (3–5).
Concern about neurotoxicity is raised when a prolonged effect on neural transmission or nociception is observed. In addition, concentration-dependent disruption of histologic structures has been demonstrated for most local anesthetics (18,19); however, when this occurs it is usually evident on histologic examination (20). In this study, most small myelinated axons (Aδ) and nonmyelinated (C-fibers) appeared to have a normal structural appearance. Thus, the application of 5% butamben suspension around the proximal sciatic nerve does not involve significant pathological alterations when the nerve is examined at the point of proximal application and more distal sites when compared to the contralateral nerve that received no injection. The minor ultrastructural alterations of the nonmyelinated axons (the extreme range of size and a paucity of the cytoskeleton) have been observed in both treated and control nerves after topical application of corticosteroid to the rat plantar nerve or Nd:YAG laser irradiation of the rat peroneal nerve, and may primarily reflect variable geometries in normal unmyelinated axons (21–23). Importantly, the isolated examples of “suggestive pathology” were also found in the contralateral noninjection samples. In the two cases where more extensive pathology and recovery were observed (one butamben 10 day, one saline 28 day), it is possible that a needle stick injury occurred at the proximal site. However, a direct effect of the butamben cannot be excluded.
Although no previous study has examined the neurotoxicity of 5% butamben suspension, reports of the histopathology of 10% butamben suspensions have produced varying results. When administered as a peripheral nerve injection in dogs, no neuropathological changes were identified on light microscopic examination (24). However, mild pathomorphological inflammatory changes and Wallerian type degeneration in dorsal spinal nerve roots was observed when 10% butamben suspension was administered in the epidural space (24). In contrast, no significant spinal cord or nerve root pathology was observed by other investigators after epidural injection (25).
The histopathologic effect of epidurally-administered 10% butamben suspension has also been reported in two studies of terminally ill cancer pain patients (3,4). In one study six patients were examined for histopathological changes after death 16–71 days after butamben injection. No patients exhibited abnormalities in the spinal nerve roots or spinal cord. Five patients showed no abnormalities in spinal ganglia, whereas one patient showed signs of atrophy and degeneration, although this was not considered to be related to butamben because it is seen occasionally at autopsy in patients who did not receive butamben. Focal necrosis was seen in the outer one-half to one-third of the dura, but all of these patients had received opioids via an indwelling epidural catheter before the butamben injection. This finding is commonly observed in patients with prior chronic epidural catheter placement (4). In another report, no significant histologic abnormalities were observed in autopsies of two patients (25).
The limitations of the current study include the use of a single dose of butamben and the nonexamination of nerve fibers proximal to the site of injection at the level of the spinal cord or dorsal root ganglia. The dose used in this study was determined as a volume that would give an adequate mass of particles to completely surround the nerve and an amount of drug considered sufficient to show any potential neurolytic effect. Dorsal spinal histology was not examined, as it was our assumption that any axonal damage that occurred previously at the proximal site would be evident as Wallerian degeneration at the more distal nerve branches. Nevertheless, the findings of this study are consistent with the clinical experience in chronic pain patients using a 5% and 10% butamben suspension who exhibit pain relief without motor or sensory impairment. Butamben suspensions seem to produce a nonneurolytic differential block of long duration.
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