Local anesthetics are widely used clinically to reversibly interrupt neuronal conduction by blocking sodium channels on the neurite membrane. However, clinical experience and laboratory examination suggest the possibility that local anesthetics have permanent neurotoxic effects when applied to peripheral neurons in large concentrations or for a long duration (1,2). Several studies conducted on mature neurons have identified the critical duration and concentration of local anesthetics required to provoke neurotoxic effects in the experimental systems (3,4). The neurotoxic effects induced by direct application of local anesthetics to immature neurons are less well understood.
Continuous application of local anesthetics using infusions, patches, or sustained release preparations, in the management of pain, exposes nerves and nerve endings to local anesthetics for a prolonged period, which may increase the risk of neurotoxicity (5). Local anesthetics are also applied repeatedly or continuously to sites where peripheral nerves may be growing or regenerating (e.g., after injury). Local anesthetics are also frequently used in pediatric surgery patients, where nerves may still be growing. Several recent studies demonstrated that a classical local anesthetic, cocaine, produces developmental neurotoxicity in vivo (6). The probability of neurotoxic effects of local anesthetics on the immature growing or regenerating neurons requires evaluation.
When neurons are growing or regenerating after injury, the nerve growth cone plays an important role in path finding and in establishing the cytoarchitecture (7). Several agents, including lysophosphatidic acid, cholinesterase inhibitors, and local anesthetics, induce growth cone collapse and interrupt nerve growth (8–10). In our previous studies, we demonstrated that the growth cone collapse induced by local anesthetics depended on the concentration and duration of exposure (10). The concentration at which tetracaine induced growth cone collapse was smaller than that at which cytoplasmic membrane function was destroyed. Furthermore, in dorsal root ganglion (DRG) neurons, only a small percentage of growth cones collapsed at a concentration <100 μM. The effects of local anesthetics on nerve growth rate have never been studied. It is possible that some cellular functions crucial for neurite extension are affected by local anesthetics administered at a small concentration.
In this study, we examined whether the growth rate of neurites was inhibited by continuous exposure to a small concentration of tetracaine that caused minimal growth cone collapse. We observed growth cone morphological changes microscopically, counted the number of filopodia, and measured the two-dimensional area of spread of the growth cone on a culture plate. For a biochemical analysis of the effect of local anesthetics, local contents of cytoskeletal proteins were examined by Western blot. In addition, we examined whether washing out tetracaine after continuous exposure resulted in a recovery of the neurite growth rate.
After approval by the Institutional Animal Care Committee, chick neural tissues were isolated from 10-d-old embryos. Peripheral neurons were prepared by dissecting DRG from lumbar paravertebral sites. After cutting off the original neurites, the tissues were plated on laminin-coated cover slips and cultured in a F-12 medium supplemented as in the method of Bottenstein et al. (11), containing 100 μg/mL of bovine pituitary extract, 2 mM of glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 20 ng/mL of mouse 7S nerve growth factor. Cultures were maintained at 37°C in an environment of 5% CO2.
Tetracaine hydrochloride was purchased from Sigma Company Ltd., (St. Louis, MO). Tetracaine was prepared in prewarmed, fresh culture media and was gently added to the cultures after 20 h in culture. The volume of the added tetracaine solution was .01 of the total volume of the culture media. The cultured neurons were examined for neurite length after exposure for 24 and 48 h to the different concentrations of tetracaine (0.1, 0.25, 0.5, 1, 2.5, 5, 10, and 20 μM). Cell viability was examined by exposing the cells to a vehicle solution for identical durations. In the experiment in which the effect of washout was examined, the tissues were kept in an incubator for 24 h after the addition of tetracaine, after which the media were gently replaced twice with fresh, prewarmed media that did not contain tetracaine.
The tissues were viewed with a 4× or 20× phase objective using a microscope. By viewing pictures taken with 4× phases, we measured neurite length from the proximal end to the distal end of the neurites (at the top of the growth cone). Growth cones at the periphery of explants were scored for collapse assay, provided they were not in contact or close proximity with other growth cones or neurites. Fifty growth cones were viewed and scored on each cover slip. Growth cones without filopodia and lamellipodia were considered to be collapsed (12). In each assay, we counted the number of filopodia and measured the two-dimensional area of the growth cone on a picture taken with 20× phases. The observation area was most distant from the site of tetracaine application.
The data obtained by six independent measurements are expressed as mean ± sd. Neurite length, percentage of growth cones showing collapse, number of filopodia, area of growth cone, and washout effect study were analyzed by two-way analysis of variance with the Scheffé test using Stat view 5.0 Software (Abasus Coop., Berkeley, CA). P values <0.05 were considered significant.
The DRGs were divided into 2 parts—neurites and cell bodies—on ice after 48 h of exposure to tetracaine (5, 10, and 20 μM and vehicle). Fifty DRGs were used for each concentration. For Western blot analysis, each part was centrifuged at 10,000 × g for 3 min. The supernatant was aspirated off, and the pellets were homogenized (using glass-on-glass homogenizer) in 100 μL of ice-cold buffer (10 mM of Tris HCl with a pH value of 7.40; 5 mM of EDTA). Fifty microliters ofthe homogenate was dissolved in 50 μL of sodium dodecylsulfate–polyacrylamide gel electrophoresis sample buffer containing 0.1 M of Tris HCl with a pH value of 7.40; 0.14 M of sodium dodecyl sulfate; 12% [vol/vol] 2-mercaptoethanol, 20% [vol/vol] glycerol, and 0.15 mM of bromophenol blue. The samples were incubated at 90°C for 3 min.
The remaining 50 μL of homogenates were used for total protein analysis, with the total protein content being measured by the method of Bradford (Bio-rad protein assay; Hercules, CA). These samples (0.83 μg of protein) were subjected to sodium dodecyl sulfate (12.5%) with polyacrylamide gel electrophoresis. The separated proteins were transblotted to polyvinylidene fluoride membrane for 30 min at 170 mA. After transfer, the membranes were blocked with 20 mM of Tris HCl with a pH value of 7.5, 500 mM of NaCl, and 0.05% Tween 20 (T-TBS) containing 5% dry milk for 2 h at room temperature. The blots were incubated overnight at 4°C with primary antibodies: anti-actin (MP Biomedicals, OH), antitubulin (Progen Immuno-Diagnostika, Heidelberg, Germany), and antineurofilament (American Research Products, Belmont) in T-TBS (1:400; actin, 1:20; tubulin dilution, and 1:20; neurofilament). After washing off the T-TBS, the appropriate secondary biotinated anti-mouse immunoglobulin (Ig)-G antibodies (ZYMED Laboratories, San Francisco, CA), or biotinated anti-mouse Ig-M antibodies (CHEMICON International, Temecula), were added in 1:100,000 dilution of T-TBS, and the samples were incubated for 60 min at room temperature. After washing T-TBS, streptavidin and biotinated alkaline phosphatase complex (Bio-rad) were added, and the samples were once again incubated for 60 min at room temperature. A color image was developed by alkaline phosphatase substrate and 5-bromo-4-chloro-3-indolyl phosphatase-nitro blue tetrazolium chloride (color developed solution, Bio-rad). The band volume was determined by Basis-Quantifire soft ware (Bio Image, Jackson, FL).
Neurite growth, measured 24 and 48 h after tetracaine application, was delayed significantly when the tetracaine concentration exceeded 5 μM as compared with vehicle- treated neurites (Figs. 1 and 2). This effect on neurite growth was not significantly different among the three concentrations of tetracaine used (5, 10, and 20 μM) both 24 and 48 h after exposure. In the experiments where the effect of washout of 5, 10, or 20 μM of tetracaine was examined, neurite growth rate of the washout group was not significantly different from the rate in the nonwashout group or the reapplication group (the same concentration of tetracaine was reapplied after the washout to examine the effect of medium exchange) 48 h after tetracaine application (Fig. 3A–C).
Growth cone collapse was assessed after the application of 5, 10, and 20 μM of tetracaine. At 48 h after tetracaine application, the growth cones collapsed by 21% ± 8% at 5 μM of tetracaine, 23% ± 10% at 10 μM of tetracaine, and 26% ± 11% at 20 μM of tetracaine (mean ± sd). These percentages of growth cone collapse were significantly increased in comparison with preapplication values and the value of vehicle-treated cells (Fig. 4).
Most of the intact neurites had growth cones with lamellipodia and filopodia at their leading edges before the application of tetracaine. With the application of tetracaine to the culture media, filopodia of growth cones retracted, and their number decreased. The number of filopodia was significantly decreased at 48 h after application of 10 and 20 μM of tetracaine (Fig. 5). The two-dimensional area of growth cones was reduced at 24 h after tetracaine application. At 48 h after the application of tetracaine, the area of growth cones reexpanded because of growth cone swelling. These changes after the application of tetracaine (0.1–20 μM) however, were not statistically significantly different from preexposure values (data not shown).
At 48 h after tetracaine application, the actin content in the tetracaine-exposed cell bodies was larger than the value in the vehicle-treated cell bodies. In contrast, actin content in the tetracaine-exposed neurites (including the axon and growth cone) was smaller than the value in the vehicle-treated neurites (Fig. 6).
In both neurites and cell bodies, the tubulin content was not significantly affected by the exposure to tetracaine (data not shown). Likewise, neurofilament content in neurites and cell bodies was not significantly affected by exposure to tetracaine (data not shown).
In the present study, we examined whether neurite growth was delayed by application of a small concentration of tetracaine. In our previous study, DRG growth cones collapsed significantly after exposure to tetracaine at a concentration of 100 μM–10 mM for 48 hours (10). Therefore, in this study, we examined a tetracaine concentration <10 μM. We found that neurite growth was significantly delayed by exposure to a tetracaine concentration larger than 5 μM. At this concentration, growth cones collapsed by 20%–30% 48 hours after application of tetracaine. Because the appearance of growth cones was mostly unaltered, we concluded that inhibition of neurite growth rate was not caused by complete collapse of growth cones or by neuronal death. We speculated that at these concentrations, growth cones are not totally collapsed, but minute morphological and motility changes may have occurred. Filopodia regulate the direction of growth cone extension and neurite elongation (13). We counted filopodia and measured the two-dimensional area of the growth cone cultures as an indication of growth cone activity (14). The number of filopodia decreased significantly after application of 10 and 20 μM of tetracaine, suggesting that growth cone morphology was influenced slightly but significantly by tetracaine.
With regard to the origin of the decrease in filopodia number, it is possible that the amount of actin in growth cones was reduced. The morphology and motility of growth cones, including filopodia, is supported by a motile cytoskeleton consisting mainly of microfilaments (15). The microfilament is composed of actin that undergoes an enzymatic polymerization (14). Although Kitagawa et al. (16) reported that neurons are irreversibly injured by the detergent properties of local anesthetics, delayed nerve growth in the present study was probably independent of the membrane lysis action of local anesthetics because the detergent properties of local anesthetics become apparent only when the concentration of local anesthetics is larger than 34.5 mM. The concentration of tetracaine in the present study was very small (<20 μM), and growth cone morphology was mostly intact, making the above explanation by Kitagawa et al. (16), with regard to neuronal growth retardation, unlikely in this study.
Several studies reported that axonal transport was inhibited by lidocaine (17). Kanai et al. (18) demonstrated that lidocaine-induced inhibition of axonal transport resulted from an accumulation of calcium ions inside the cell. In our previous study, disruption of intracellular calcium regulation was observed after the application of tetracaine to the cultured growing neurons (19). In the present study, the amount of actin in cell bodies increased, whereas actin in growth cones and neurites decreased. These results suggest that axonal transport and trafficking of actin were partly inhibited at a small concentration of tetracaine and that inhibition of axonal transport may be related to slow nerve growth and the change of growth cone morphology.
By using another type of cell, Bengtsson et al. (20) reported that tetracaine enhanced accumulation of F-actin in the periphery of fMet-Leu-Phe-stimulated neutrophils. Actin assembly may be affected by tetracaine. In our present study, the intracellular actin shift may have been caused by similar intracellular mechanisms. Further studies that directly measure the effect of local anesthetics on axonal transport are required to further elucidate the exact mechanism of this phenomenon.
The sizes of the growth cones after tetracaine application were not significantly different from the preexposure values. Although the number of filopodia was reduced, part of the growth cone appeared to be swollen after exposure to tetracaine. Thus, the net size of the growth cones was maintained even after retraction of the filopodia. It is possible that membrane permeability is increased by local anesthetics (21). Increase in intracellular calcium might be related to growth cone swelling and inhibition of neurite growth rate (22). Tsuda et al. (23) reported that tetracaine inhibits the actomyosin motility in vitro. Pierzchalska et al. (24) demonstrated that tetracaine affects the actomyosin system along with changes in morphology of human skin fibroblasts. Considering these results, it is possible that morphological changes of growth cones were not induced solely by a reduction of the actin quantity. Tetracaine may directly or indirectly inhibit the cytoskeletal system of growth cones.
In the present study, the delay in neurite growth induced by a small concentration of tetracaine did not recover by washing out the tetracaine. Dysfunction of some intragrowth cone enzymes may have continued after the temporary exposure to tetracaine. In our previous study, growth cone collapse induced by 1 mM of tetracaine was irreversible and did not recover by washing out the tetracaine (10). However, we could not follow the growth of neurites for longer than 48 hours after tetracaine washout. The viability of neurons declined after 48 hours in our cultures, probably because of the mechanical effects of medium exchange, which detaches many neurites from the floor of the culture plate. Thus, it is possible that the neurites slowly recover their rate of growth after the 48-hour observation period.
In our previous study, we examined the neurotoxicity of tetracaine at large concentrations. In the present study, we investigated the effects of tetracaine at smaller concentrations. In clinical settings, other local anesthetics, such as lidocaine, bupivacaine, and ropivacaine, are more often used. Effects of such anesthetics should be examined in future studies to clarify the clinical significance of the developmental neurotoxicity of local anesthetics.
In summary, neurite growth was delayed by a small concentration of tetracaine, whereas growth cone morphology remained unaffected. This effect may be accompanied by a cytoskeletal change and filopodia retraction in growth cones. Washout of tetracaine did not result in a recovery from these effects. Therefore, although the present data obtained from in vitro experiments with avian neurons cannot be directly applied to clinical cases, local anesthetics should be used carefully on growing or regenerating neural tissues, particularly after long exposures.
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