Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell death. The authors therefore investigated in vitro (cultured primary sensory neurons) and in vivo (sciatic nerve block model) the potential neuroprotective effect of the p38 MAPK inhibitor SB203580 administered together with a clinical (lidocaine) or investigational (amitriptyline) local anesthetic.


Cell survival and mitochondrial depolarization as marker of apoptotic cell death was assessed in rat dorsal root ganglia incubated with lidocaine or amitriptyline either with or without the addition of SB203580. Similarly, in a sciatic nerve block model, the authors assessed wallerian degeneration by light microscopy to detect a potential mitigating effect of MAPK inhibition.


Lidocaine at 40 mm/approximately 1% and amitriptyline at 100 microm reduce neuron count, but coincubation with the p38 MAPK inhibitor SB203580 at 10 mum significantly reduces cytotoxicity and the number of neurons exhibiting mitochondrial depolarization. Also, wallerian degeneration and demyelination induced by lidocaine (600 mm/approximately 15%) and amitriptyline (10 mm/approximately 0.3%) seem to be mitigated by SB203580.


The cytotoxic effect of lidocaine and amitriptyline in cultured dorsal root ganglia cells and the nerve degeneration in the rat sciatic nerve model seem, at least in part, to be mediated by apoptosis but seem efficiently blocked by an inhibitor of p38 MAPK, making it conceivable that coinjection might be useful in preventing local anesthetic-induced neurotoxicity.

DEVELOPMENT of new local anesthetics (LAs) is seriously hampered by neurotoxicity. Recently, several new promising LAs under development by our group1and other investigators2,3had reached clinical trials that had to be halted because of presumed neurotoxicity. In addition, LAs in current clinical use are well known to cause direct neuronal toxicity, i.e. , transient neurologic injury,4–8cauda equina syndrome,9–11and even permanent loss of spinal cord function.12Other than using the lowest possible concentration/dosage of LAs, no other option is currently available to prevent LA-induced direct neurotoxicity.

Although severe LA-induced injury kills neuronal cells via  necrosis, increasing evidence suggests that, as severity decreases, apoptosis becomes the main mechanism of cell death.13,14Interestingly, it recently was shown that LAs, even at clinical concentrations, induce activation of the p38 mitogen-activated protein kinase (MAPK) system,15,16a specific intracellular signaling pathway also involved in apoptosis.

Mitogen-activated protein kinases are a family of protein kinases that phosphorylate specific serines and threonines of target protein substrates and regulate wide-ranging cellular activities such as gene expression, mitosis, movement, and metabolism, as well as apoptosis. The subfamily of p38 MAPKs is activated by many stimuli, including inflammatory cytokines (e.g. , tumor necrosis factor α)17–19and Ca2+,20–22both of which are involved in LA toxicity. Although apoptosis is brought about by an extremely complex system, many antiapoptotic drugs are available (some of which are in phase II clinical trials23–25), raising the possibility that coinjection of LAs with drugs inhibiting apoptosis might be one approach to decreasing neurotoxicity. We selected lidocaine26as a representative LA in clinical use and amitriptyline as a representative investigational LA, both of which have a high potential for neurotoxicity, particularly amitriptyline.27 

We tested the hypothesis that inhibition of the p38 MAPK reduces neurotoxicity caused by the LAs lidocaine and amitriptyline by inhibiting apoptosis. Specifically, we (1) incubated cultivated dorsal root ganglia cells with (a) lidocaine alone or in combination with the p38 MAPK inhibitor SB203580 and (b) amitriptyline alone or in combination with SB203580, assessing cell survival and apoptosis (via  detection of mitochondrial depolarization), and (2) injected, in a sciatic nerve block model, (a) lidocaine alone or in combination with SB203580 and (b) amitriptyline alone or in combination with the SB203580 and assessed the extent of wallerian degeneration.

We used the model of dissociated rat dorsal root ganglion (DRG) culture for evaluation of cytotoxicity by lidocaine or amitriptyline and the potential alleviation thereof by a p38 MAPK inhibitor. To assess the mechanism of toxicity, we used an early marker of apoptosis, i.e. , mitochondrial depolarization. We evaluated the extent of sciatic nerve degeneration by light microscopy after perineural drug injection. We selected the subfascial sciatic nerve injection approach, because this allows application of drug directly onto the nerve with visual confirmation and therefore yields the most reproducible results. The concentrations of test drugs were chosen on the basis of preliminary studies showing that this concentration reduces the cell count by approximately 50% or elicits substantial neural damage, such that a protective effect of p38 MAPK inhibition would be most clearly visible.


Unless stated otherwise, drugs were purchased from Sigma Aldrich (Vienna, Austria, or St. Louis, MO). For the in vitro  experiments, the pH of the stock solutions of 1 m lidocaine was 4.65, and that for 1 mm amitriptyline was 4.46 (in dimethyl sulfoxide [DMSO]). Because a larger volume of amitriptyline stock solution had to be added to the medium (pH of 7.4), the pH of this final solution was lower for amitriptyline than for lidocaine (pH 7.1 for 100 μm amitriptyline and pH 7.38 for 40 mm lidocaine). The osmolality of the medium incubated with lidocaine or amitriptyline was not significantly higher than that of control cultures incubated with vehicle only. The concentration of DMSO was approximately 0.1%, which is known to be the threshold for neurotoxicity.28Because a larger volume of amitriptyline than lidocaine stock solution (which contains the potentially neurotoxic substance DMSO) had to be added to the medium to yield the desired final concentration, we increased the DMSO content of the lidocaine solution to obtain identical DMSO concentrations. Similarly, for control cultures with vehicle only (medium), the same concentration of DMSO was used. Although the concentration of DMSO was approximately 0.1%, we thus could obtain identical conditions for all treatment groups to avoid a potentially confounding variable.

For the in vivo  experiments, 15% lidocaine and 10 mm amitriptyline were freshly prepared by dissolving them in normal saline, resulting in pH values of 4.8 and 6.20, respectively. The addition of SB253080 did not change the pH significantly. We did not adjust the pH, because buffering by the tissue fluid, which has a pH of 7.4, is rapid.

In Vitro  Experiments

Neuron Culture.

Dorsal root ganglion cultures were obtained in a manner similar to that described previously.20,29Briefly, neurons were acutely harvested from adult (8–9 weeks) female Sprague-Dawley rats, which were killed by carbon dioxide narcosis according to the institutional protocol (Animal Committee of the Austrian Federal Ministry of Education, Science and Culture, Vienna, Austria). DRG were desheathed and incubated in 5,000 U/ml collagenase for 90 min at 37°C, followed by 15 min in 0.25% trypsin–EDTA. After dissociation in Roswell Park Memorial Institute (RPMI) medium containing 10% horse–5% fetal bovine serum, neurons were plated in RPMI medium supplemented with nitrogen additives (1:100) and antibiotics (penicillin, 1,000 U/ml; streptomycin, 1,000 μg/ml; and amphotericin B, 25 μg/ml in 0.85% saline), all purchased from Invitrogen (Vienna, Austria).

Neurons were allowed for 24 h to adhere to the glass floor of dishes coated with poly-d-lysine/laminin. Poly-d-lysine was applied at a concentration of 0.1 mg/ml in distilled water and laminin at 7 μg/ml in RPMI solution. Cell cultures were kept at 37°C in a humidified atmosphere containing 5% CO2.

Assessment of Cell Survival.

To confirm dose-dependent toxicity of lidocaine and amitriptyline and to assess whether inhibition of p38 MAPK attenuated cytotoxicity, we incubated neurons with 50, 100, and 150 μm amitriptyline or 20, 40, and 60 mm lidocaine, either with or without the addition of 1 or 10 μm SB203580, a selective pyridinyl imidazole inhibitor of p38 MAPK. After 24 h, cultures were fixed with paraformaldehyde (4%) and evaluated by the same experimenter with regard to survival by determining total cell counts. Surviving neurons were defined as cells with round cell bodies, a clearly visible nucleus, and neurofilament-positive staining distinguishing them from glial cells. To confirm that a decreased number of cells is indeed due to cell death (vs.  reduced adherence to the glass cover slip), we performed pilot studies examining all cells in several culture dishes indicating that cells not counted as “survived” actually had no visible nucleus and deformed cell bodies. In each dish, neurons were counted in 50 visual fields (20× magnification). No differentiation of cell sizes was made.


In a different experiment, we determined the incidence of mitochondrial depolarization, an early hallmark of apoptosis,30after exposure to lidocaine, amitriptyline, or control medium. In addition, we assessed whether inhibition of apoptosis is the mechanism by which the p38 MAPK inhibitor SB203580 mitigates LA-induced neurotoxicity. Therefore, we used the fluorescent cell-permeant dye JC-1, which exists either as an aggregate at “physiologic” membrane potentials (ΔΨm less than −100 mV, red fluorescence, 590 nm) or as a monomer at depolarized membrane potentials greater than −100 mV (green fluorescence, 527 nm). JC-1 is selectively incorporated into the mitochondrial membrane and changes its fluorescent color from red to green upon mitochondrial depolarization, suggesting that the ratio of green/red (527/590 nm) fluorescence can be used as an indicator of mitochondrial membrane potential ΔΨm.31,32 

We compared alterations in ΔΨm of control neurons incubated with vehicle only with cultures treated with 100 μm amitriptyline or 40 mm lidocaine. In addition, cultures were incubated with the inhibitor of p38 MAPK, SB203580, at 10 μm. After 4 h of incubation, neurons were incubated with 15 μm JC-1 for 50 min at 37°C. For that purpose, the JC-1 dye was freshly prepared by diluting a stock solution (1 mg/ml) 1:100 in RPMI medium. Then the cultures were washed twice with RPMI medium supplemented with nitrogen additives to remove excess dye. Cell cultures were evaluated for the number of neurons exhibiting mitochondrial depolarization and the average ratio of green and red fluorescence.

In Vivo  Experiments

Subfascial Sciatic Nerve Injection.

The protocol for animal experimentation was reviewed and approved by the Harvard Medical Area Standing Committee on Animals (Boston, Massachusetts). For a pilot study, we selected 0.2 ml lidocaine, at a concentration of 15%/approximately 600 mm, alone or in combination with 10 μm SB203580 (n = 6/group), to be injected directly beneath the clear fascia surrounding the nerve but outside the perineurium, proximal to the sciatic bifurcation. Amitriptyline at 10 mm/approximately 0.3% alone or in combination with 10 μm SB203580 (n = 8/group) was used for comparison. Potential toxicity of either the MAPK inhibitor or normal saline alone was assessed by injecting four rats with SB203580 or four with normal saline alone.

With animals under 1–2% isoflurane inhalation anesthesia, the sciatic nerves of adult female Sprague-Dawley rats weighing approximately 200 g were exposed by lateral incision of the thighs and division of the superficial fascia and muscle as described previously.33The superficial muscle layer was sutured with 4-0 silk, and the wound was closed with metal clips. After the animals recovered from general anesthesia, their sciatic nerve function was evaluated grossly by reaction to pinch of the fifth toe with a forceps and observed for flaccid paralysis due to motor blockade. The contralateral side was used as the control. Animals were then tested in the same manner daily until killed on day 7 after injection.

Pathologic Evaluation.

We excised the sciatic nerves following the anesthesia protocol used for surgery 7 days after administering the test dose; the rats were then killed with an intraperitoneal injection of sodium pentobarbital (70 mg/kg). For fixation, the nerves, measuring approximately 2 cm long with the injection site in the middle, were placed on a wooden stick and immersed in 2.5% phosphate-buffered glutaraldehyde for 24 h. These were rinsed three times with phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in serial concentrations of alcohol, and embedded in araldite according to the recommended procedure for neurotoxicologic tissue evaluation.34Twenty 1-μm-thick semithin sections from the central 2-mm block of each 6-mm-long segment were prepared for light microscopy and stained with methylene blue, azure II. An observer unaware of the experimental groupings evaluated the tissue sections. A value was assigned for each histologic slide based on a scale with 1-unit increments from 0 to 8 in which 0 represented a normal nerve and 8 represented a finding of wallerian degeneration extending throughout the nerve bundle.

Statistical Analyses

Analysis of variance was used to compare cell culture survival data between treatment groups (20/40/60 mm lidocaine, lidocaine + 1 μm SB203580, lidocaine + 10 μm SB203580; and 50/100/150 μm amitriptyline, amitriptyline + 1 μm SB203580, amitriptyline + 10 μm SB203580) relative to control (vehicle), with the post hoc  Dunnett t  test method applied to evaluate group differences and to account for multiple comparisons. Analysis of variance with the Bonferroni post hoc  correction was used to compare results from mitochondrial depolarization experiments. Means ± SD were used to summarize the results, because the cell counts followed a normal distribution as tested by the Wilk-Shapiro test. Two-tailed values of P < 0.05 were considered statistically significant. A power analysis was performed for the cell culture experiments and indicated that the sample size of five cultures per treatment group would provide 80% power (β= 0.20) for detecting a difference of 30% or more in an average cell count assuming an SD of 10% within a group (version 5.0, nQuery Advisor; Statistical Solutions, Cork, Ireland). The rationale for choosing a 10% SD is based primarily on the analysis of preliminary data, which indicated that the within-group variability was tight and suggested less variability in the cell culture experiments for a given treatment (lidocaine or amitriptyline, with or without SB203580) as compared with control, specifically with respect to data on cell survival. We chose to specify a 10% SD for the power analyses to yield a relevant effect size (albeit a large effect size of 3.0) estimate for detecting group mean differences by analysis of variance. Furthermore, this SD of 10% was considered a reasonable, scientific estimate of variability.

Injury scores were tested by the Mann–Whitney U test, because skewness was detected, and results are therefore given as the median (range). Because the in vivo  experiments were intended as a preliminary study only, we did not perform any power analysis.

In Vitro  Experiments

Lidocaine Reduces Cell Count, and Coincubation with SB203580 Significantly Reduces Lidocaine Cytotoxicity.

Incubation of DRG cultures with lidocaine at 40 mm decreased the average neuron number in cultures from 506 ± 20 to 228 ± 50 (survival of 45%), and coincubation with SB203580 at 1 μm did not significantly prevent neuronal death (cell count of 246 ± 50). However, at a concentration of 10 μm SB203580, the neuron count was 411 ± 48 (survival rate of 81%; P < 0.001) and, as with amitriptyline, not significantly lower (P > 0.05) than that of controls (medium only) (fig. 1A). The salvaging effect of 10 μm SB203580 was also apparent for 20 and 60 mm lidocaine. At these concentrations, cell survival was increased from 47 ± 5 to 57 ± 20% (not significant) and 30 ± 12 to 58 ± 3% (P < 0.01), respectively. Incubation with SB203580 alone had no detectable effect.

Amitriptyline Reduces Cell Count, and Coincubation with the p38 MAPK Inhibitor SB203580 Significantly Reduces Amitriptyline Cytotoxicity.

We confirmed a cytotoxic effect in neurons exposed to amitriptyline at a concentration of 100 μm; the average neuron count as compared with controls (444 ± 42) significantly decreased in cultures incubated with amitriptyline at 100 μm (survival of 38%, 169 ± 20; P < 0.001). Addition of 1 μm SB203580 did not significantly attenuate this effect (average cell count 213 ± 37). In contrast, coincubation with 10 μm SB203580 alleviated the decrease in neuron count, with the survival rate reaching 82% (average cell count 365 ± 30) (fig. 1B). The latter survival rate was significantly higher than that of cultures incubated with 100 μm amitriptyline alone (P < 0.001), although not significantly lower than that of controls (P > 0.05). Improved survival in cultures coincubated with 10 μm SB203580 was also detectable at 50 μm and 150 μm amitriptyline. At these concentrations, cell survival was increased from 43 ± 6 to 64 ± 4% (P < 0.01) and 32 ± 10 to 62 ± 18% (P < 0.01), respectively.

Lidocaine and Amitriptyline Cause Mitochondrial Depolarization, and Coincubation with SB203580 Leads to Reduction Thereof via  Inhibition of p38 MAPK.

Figure 2displays representative cells demonstrating loss of membrane potential after exposure to lidocaine or amitriptyline and the prevention of this loss by coincubation with 10 μm SB203580. The percentage of control neurons incubated only with medium (n = 56) displaying depolarized ΔΨm was 7.2 ± 3.0%, indistinguishable from the percentage in neurons coincubated with only SB203580. Incubation with 40 mm lidocaine (fig. 3A) resulted in 57.1 ± 8.6% of neurons with depolarized ΔΨm (n = 53; P < 0.001), and addition of SB203580 decreased this to 34.6 ± 9.4% (n = 52; P < 0.05 compared with controls; P < 0.05 compared with lidocaine group). Similarly, incubation with amitriptyline (fig. 3B) resulted in 20.9 ± 3.8% of neurons with depolarized ΔΨm (n = 48; P < 0.01), whereas coincubation with SB203580 reduced this to 7.8 ± 3.5% (n = 52; not significant compared with controls; P < 0.05 compared with the amitriptyline group).

The average ratio of fluorescent green/red neurons in control cultures was 0.31 ± 0.42, indicating a physiologic mitochondrial membrane potential. Incubation with lidocaine (fig. 3C) increased this ratio to 1.73 ± 1.58 (P < 0.001), whereas coadministration of SB203580 resulted in a ratio of 0.75 ± 0.67 (not significant compared with controls; P < 0.001 compared with the lidocaine group). Treatment with amitriptyline (fig. 3D) increased the fluorescent ratio to 0.60 ± 0.70 (P < 0.001), and coincubation with SB203580 resulted in a ratio of 0.20 ± 0.29 (not significant compared with controls; P < 0.01 compared with amitriptyline group).

In Vivo  Experiments

Sciatic Nerve Degeneration Is Induced by Lidocaine and Mitigated by SB203580.

Two rats from the lidocaine group were excluded, because they had wound dehiscence and gross infection and were killed per protocol. All of the rats remaining for histologic analyses (15% lidocaine: n = 5; 15% lidocaine combined with SB203580: n = 5) demonstrated complete block of motor and sensory sciatic nerve functions but recovered completely by the next day. Rats treated with 15% lidocaine alone demonstrated severe histologic changes (i.e. , wallerian degeneration), with an injury score of 6 (5–8) (representative sample in fig. 4A).

Addition of SB203580 reduced these findings of wallerian degeneration characteristic of LA-induced neurotoxicity, with an injury score of 3 (1–6), P = 0.15 (representative sample in fig. 4B). Histologic findings were normal in rats treated with 10 μm SB203580 or normal saline alone (n = 4/group) (representative sample not shown).

Sciatic Nerve Degeneration Is Induced by Amitriptyline and Mitigated by SB203580.

Similar to the lidocaine-treated animals, all rats treated with amitriptyline developed complete sciatic nerve block, as indicated by failure to respond to pinch of the fifth toe as well as by flaccid paralysis. No overt neurologic deficit was seen after 24 h, i.e. , pinch to the fifth toe was followed by a brisk withdrawal reaction and vocalization identical to that of the control (contralateral limb). Similarly, posture, gait, and walking pattern were unaffected. Nevertheless, upon histologic analyses, degenerative changes of sciatic nerves treated with amitriptyline (fig. 5A) and effective reduction of those changes in nerves treated with amitriptyline combined with MAPK inhibitor (fig. 5B) were observed. However, similar to the group treated with lidocaine, those treated with amitriptyline combined with SB203580 had markedly improved scores, but differences were not statistically significant.

We have shown in vitro  (cell culture model) and in vivo  (sciatic nerve injection model) that a p38 MAPK inhibitor is capable of mitigating clinical (lidocaine) and investigational (amitriptyline) LA neurotoxicity, as suggested by increased neuron survival and improved histologic evaluation (i.e. , less wallerian degeneration), respectively. A significant decrease in the percentage of cells demonstrating loss of mitochondrial membrane potential after coincubation with SB203580 compared with that of cells incubated with lidocaine or amitriptyline alone suggests apoptosis as a mechanism of injury induced by these two LA agents.

Apoptosis, probably initiated by LA-induced p38 MAPK activation, leads to mitochondrial membrane permeabilization, which causes the release of cytochrome c  from the mitochondrial intermembrane space, marking the point of no return in the process of cell death. Once in the cytosol, cytochrome c  triggers the assembly of a caspase activation complex, leading to activation of caspase-3, which in turn triggers oligonucleosomal DNA fragmentation, the hallmark of apoptosis.35–42We demonstrated that both lidocaine and amitriptyline elicit mitochondrial depolarization, an indirect marker of apoptosis. Mitochondrial membrane depolarization is effectively observed during apoptosis; however, it is not specific of apoptosis (some drugs, including LAs at high concentration, uncouple oxidative phosphorylation with a decrease in membrane potential43). Nevertheless, our results corroborate previous investigations in neuronal cell lines suggesting that apoptosis is, at least in part, responsible for the phenomenon of LA-induced nerve injury.15,44,45 

Because the apoptotic process is to some extent reversible, the use of neuroprotective drugs interacting with apoptotic pathways could be of considerable benefit. In addition, p38 MAPK has been described as mediating various neurodegenerative pathways in primary sensory neurons,46,47while its inhibition has been shown to enhance neuronal survival.48Therefore, coapplication of drugs such as p38 MAPK inhibitors may be useful for minimizing the neurotoxic effects of both conventional and novel LAs.

For the in vitro  experiments, we selected drug concentrations to achieve approximately 50% cell death, so that a potential mitigating effect of cytotoxicity, i.e. , increased cell survival, is easily detectable. Although not the aim of this study, the concentrations chosen for lidocaine seem clinically relevant, because other investigators have chosen similar concentrations for cell culture experiments to simulate expected lidocaine concentrations after intrathecal application.44Similarly, we chose the incubation time of 24 h because under clinical conditions, e.g. , epidural anesthesia, primary afferent somata may be exposed to LAs for several days.

For the in vivo  experiments, however, the concentrations used clearly exceeded those used clinically. We selected these high concentrations because we set out to find a concentration at which we would definitely encounter toxicity, and preferably in all animals, to keep the number of animals needed as low as possible. As Eisenach and Yaksh49pointed out in an editorial, studies with lidocaine at 1% would require many-fold more animals. Because in pilot studies (data not shown), not even 10% lidocaine produced significant histopathologic lesions (i.e. , wallerian degeneration) in all animals, we increased the concentration to 15%. This approach is commonly undertaken in toxicity studies; otherwise an ethically unjustifiable number of animals would need to be killed.

It was beyond the scope of this study to assess whether administration of p38 MAPK inhibitors could also mitigate neurotoxicity after an LA-induced injury occurred. To the best of our knowledge, this has not yet been studied but certainly could be of great clinical relevance. Interestingly, in a rat sciatic nerve crush injury model, the peroral administration of a MAPK inhibitor (SD-169, a proprietary oral inhibitor of p38 MAPK activity not used in the current study) was shown to be effective in reducing injury, both neurobehaviorally and histologically.50For example, pinching the SD-169–treated animals revealed significantly better recovery from sensory deficit, and regenerating nerves were morphologically more mature than untreated nerves when observed 28 days after transection. Furthermore, SD-169 significantly reduced tumor necrosis factor-mediated primary Schwann cell death in culture experiments.50 

In this study, we initially observed a P  value of 0.15 for results with n = 5 animals treated with lidocaine combined with SB203580 with respect to reduction of neuronal injury. The sample size was too small to attain statistical significance, although it clearly demonstrated a beneficial trend. There was evidence from these experiments that a larger group of animals is necessary to reach significance and that small n values result in treatment effects that are almost statistically significant (considered a strong trend) but do not satisfy the a priori  definition of P < 0.05 because of low statistical power and a consequently high risk of a type II (β) error. This is clear inasmuch as evaluation of differences in injury among a group of five animals treated with lidocaine combined with SB203580 did not reach significance (P = 0.15), but combining results from an additional six animals amplified the effect (P < 0.001, Mann–Whitney U test). We applied nonparametric statistical tests because of the distribution of the data and the small sample sizes. Moreover, our results for amitriptyline showed noticeable improvements in injury results; however, again because of the small sample size, we were unable to make a definitive statistical statement. Notwithstanding, we have interpreted our results, although cautiously, as indicative of a protective neuronal advantage, although based on a limited number of animals. However, in moving ahead, we realize that a larger sample size in each LA group (including controls) is necessary (projected to be at least 10 animals/group for 80% power) to have an acceptable level of statistical power for detecting statistically significant treatment group differences and avoiding false negatives (type II or β errors).

When eliciting peripheral nerve blocks, the primary target of LA action is the axon and not the DRG cell. Although our investigation focused on in vivo  sciatic nerve toxicity (i.e. , axonal toxicity) and in vitro  DRG cells (i.e. , ganglionic toxicity), we believe there is a strong connection, because death of DRG following demyelination and axonal degeneration after peripheral nerve lesions has clearly been demonstrated to be attributable to apoptosis.51Nevertheless, future studies probably should attempt to correlate findings from peripheral nerve block models with Schwann cell cultures and spinal block models with DRG cell cultures.

In conclusion, apoptosis most probably mediates cytotoxicity in cultured DRG cells and axonal degeneration and demyelination in a rat sciatic nerve model elicited by amitriptyline and lidocaine. An inhibitor of p38 MAPK, SB203580, seems to block this process, making it conceivable that its coinjection might prevent LA-induced nerve injury. More histologic studies involving larger sample sizes and additional models (e.g. , intrathecal application) are needed to confirm our preliminary in vivo  findings.

Fridrich P, Eappen S, Jaeger W, Schernhammer E, Zizza AM, Wang GK, Gerner P: Phase Ia and Ib study of amitriptyline for ulnar nerve block in humans: Side effects and efficacy. Anesthesiology 2004; 100:1511–8
Galvez-Mugica MA, Santos-Ampuero MA, Novalbos J, Gallego SS, Galiano A, Gilsanz F, Garcia AG, Abad-Santos F: Ulnar nerve block induced by the new local anesthetic IQB-9302 in healthy volunteers: A comparison with bupivacaine. Anesth Analg 2001; 93:1316–20
Drager C, Benziger D, Gao F, Berde CB: Prolonged intercostal nerve blockade in sheep using controlled-release of bupivacaine and dexamethasone from polymer microspheres. Anesthesiology 1998; 89:969–79
Drasner K: Lidocaine spinal anesthesia: A vanishing therapeutic index? Anesthesiology 1997; 87:469–72
Drasner K: Local anesthetic neurotoxicity: Clinical injury and strategies that may minimize risk. Reg Anesth Pain Med 2002; 27:576–80
Freedman JM, Li DK, Drasner K, Jaskela MC, Larsen B, Wi S: Transient neurologic symptoms after spinal anesthesia: An epidemiologic study of 1,863 patients. Anesthesiology 1998; 89:633–41
Hampl KF, Heinzmann-Wiedmer S, Luginbuehl I, Harms C, Seeberger M, Schneider MC, Drasner K: Transient neurologic symptoms after spinal anesthesia: A lower incidence with prilocaine and bupivacaine than with lidocaine. Anesthesiology 1998; 88:629–33
Hampl KF, Schneider MC, Pargger H, Gut J, Drewe J, Drasner K: A similar incidence of transient neurologic symptoms after spinal anesthesia with 2% and 5% lidocaine. Anesth Analg 1996; 83:1051–4
Horlocker TT, McGregor DG, Matsushige DK, Chantigian RC, Schroeder DR, Besse JA: Neurologic complications of 603 consecutive continuous spinal anesthetics using macrocatheter and microcatheter techniques. Perioperative Outcomes Group. Anesth Analg 1997; 84:1063–70
Loo CC, Irestedt L: Cauda equina syndrome after spinal anaesthesia with hyperbaric 5% lignocaine: A review of six cases of cauda equina syndrome reported to the Swedish Pharmaceutical Insurance 1993-1997. Acta Anaesthesiol Scand 1999; 43:371–9
Rigler ML, Drasner K, Krejcie TC, Yelich SJ, Scholnick FT, DeFontes J, Bohner D: Cauda equina syndrome after continuous spinal anesthesia. Anesth Analg 1991; 72:275–81
Benumof JL: Permanent loss of cervical spinal cord function associated with interscalene block performed under general anesthesia. Anesthesiology 2000; 93:1541–4
Koike T, Tanaka S, Oda1 T, Ninomiya T: Sodium overload through voltage-dependent Na(+) channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro . Brain Res Bull 2000; 51:345–55
Mukherjee PK, Marcheselli VL, Serhan CN, Bazan NG: Neuroprotectin D1: A docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Natl Acad Sci U S A 2004; 101:8491–6
Johnson ME, Wang H, Uhl CB: Neuronal p38 mitogen activated protein kinase in a cell culture model of transient neurologic syndrome following lidocaine exposure (abstract). Anesthesiology 2004; 101:A1069
Tan Z, Dohi S, Chen J, Banno Y, Nozawa Y: Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death. Anesthesiology 2002; 96:1191–201
Grethe S, Ares MP, Andersson T, Porn-Ares MI: p38 MAPK mediates TNF-induced apoptosis in endothelial cells via  phosphorylation and downregulation of Bcl-x(L). Exp Cell Res 2004; 298:632–42
Ohtori S, Takahashi K, Moriya H, Myers RR: TNF-alpha and TNF-alpha receptor type 1 upregulation in glia and neurons after peripheral nerve injury: Studies in murine DRG and spinal cord. Spine 2004; 29:1082–8
Pollock J, McFarlane SM, Connell MC, Zehavi U, Vandenabeele P, MacEwan DJ, Scott RH: TNF-alpha receptors simultaneously activate Ca2+ mobilisation and stress kinases in cultured sensory neurones. Neuropharmacology 2002; 42:93–106
Gold MS, Reichling DB, Hampl KF, Drasner K, Levine JD: Lidocaine toxicity in primary afferent neurons from the rat. J Pharmacol Exp Ther 1998; 285:413–21
Sattler R, Tymianski M: Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Mol Neurobiol 2001; 24:107–29
Arundine M, Tymianski M: Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium 2003; 34:325–37
Haddad JJ: VX-745: Vertex Pharmaceuticals. Curr Opin Investig Drugs 2001; 2:1070–6
Parasrampuria DA, de Boer P, Desai-Krieger D, Chow AT, Jones CR: Single-dose pharmacokinetics and pharmacodynamics of RWJ 67657, a specific p38 mitogen-activated protein kinase inhibitor: A first-in-human study. J Clin Pharmacol 2003; 43:406–13
Fijen JW, Tulleken JE, Kobold AC, de BP, van der Werf TS, Ligtenberg JJ, Spanjersberg R, Zijlstra JG: Inhibition of p38 mitogen-activated protein kinase: Dose-dependent suppression of leukocyte and endothelial response after endotoxin challenge in humans. Crit Care Med 2002; 30:841–5
Johnson ME: Neurotoxicity of lidocaine: Implications for spinal anesthesia and neuroprotection. J Neurosurg Anesthesiol 2004; 16:80–3
Estebe JP, Myers RR: Amitriptyline neurotoxicity: Dose-related pathology after topical application to rat sciatic nerve. Anesthesiology 2004; 100:1519–25
Ginsburg K, Narahashi T: Time course and temperature dependence of allethrin modulation of sodium channels in rat dorsal root ganglion cells. Brain Res 1999; 847:38–49
Ginsburg KS, Narahashi T: Differential sensitivity of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels to the insecticide allethrin in rat dorsal root ganglion neurons. Brain Res 1993; 627:239–48
Ly JD, Grubb DR, Lawen A: The mitochondrial membrane potential (deltapsi(m)) in apoptosis: An update. Apoptosis 2003; 8:115–28
Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C: A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993; 197:40–5
Russell JW, Golovoy D, Vincent AM, Mahendru P, Olzmann JA, Mentzer A, Feldman EL: High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J 2002; 16:1738–48
Myers RR, Kalichman MW, Reisner LS, Powell HC: Neurotoxicity of local anesthetics: Altered perineurial permeability, edema, and nerve fiber injury. Anesthesiology 1986; 64:29–35
Myers RR, Sommer C: Methodology for spinal neurotoxicity studies. Reg Anesth 1993; 18:439–47
Penninger JM, Kroemer G: Mitochondria, AIF and caspases–rivaling for cell death execution. Nat Cell Biol 2003; 5:97–9
Adams JM: Ways of dying: Multiple pathways to apoptosis. Genes Dev 2003; 17:2481–95
Jordan J, Galindo MF, Gonzalez-Garcia C, Cena V: Role and regulation of p53 in depolarization-induced neuronal death. Neuroscience 2003; 122:707–15
Kroemer G, Reed JC: Mitochondrial control of cell death. Nat Med 2000; 6:513–9
Martinou JC, Green DR: Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001; 2:63–7
Newmeyer DD, Ferguson-Miller S: Mitochondria: Releasing power for life and unleashing the machineries of death. Cell 2003; 112:481–90
Zamzami N, Kroemer G: Apoptosis: Mitochondrial membrane permeabilization: The (w)hole story? Curr Biol 2003; 13:R71–3
Zamzami N, Kroemer G: The mitochondrion in apoptosis: How Pandora’s box opens. Nat Rev Mol Cell Biol 2001; 2:67–71
Sztark F, Malgat M, Dabadie P, Mazat JP: Comparison of the effects of bupivacaine and ropivacaine on heart cell mitochondrial bioenergetics. Anesthesiology 1998; 88:1340–9
Johnson ME, Uhl CB, Spittler KH, Wang H, Gores GJ: Mitochondrial injury and caspase activation by the local anesthetic lidocaine. Anesthesiology 2004; 101:1184–94
Boselli E, Duflo F, Debon R, Allaouchiche B, Chassard D, Thomas L, Portoukalian J: The induction of apoptosis by local anesthetics: A comparison between lidocaine and ropivacaine. Anesth Analg 2003; 96:755–6
Obata K, Yamanaka H, Kobayashi K, Dai Y, Mizushima T, Katsura H, Fukuoka T, Tokunaga A, Noguchi K: Role of mitogen-activated protein kinase activation in injured and intact primary afferent neurons for mechanical and heat hypersensitivity after spinal nerve ligation. J Neurosci 2004; 24:10211–22
Price SA, Agthong S, Middlemas AB, Tomlinson DR: Mitogen-activated protein kinase p38 mediates reduced nerve conduction velocity in experimental diabetic neuropathy: Interactions with aldose reductase. Diabetes 2004; 53:1851–6
Horstmann S, Kahle PJ, Borasio GD: Inhibitors of p38 mitogen-activated protein kinase promote neuronal survival in vitro . J Neurosci Res 1998; 52:483–90
Eisenach JC, Yaksh TL: Safety in numbers: How do we study toxicity of spinal analgesics? Anesthesiology 2002; 97:1047–9
Myers RR, Sekiguchi Y, Kikuchi S, Scott B, Medicherla S, Protter A, Campana WM: Inhibition of p38 MAP kinase activity enhances axonal regeneration. Exp Neurol 2003; 184:606–14
Azkue JJ, Zimmermann M, Hsieh TF, Herdegen T: Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons. Eur J Neurosci 1998; 10:2204–6