Neurotoxicity of adjuvants used in perineural anesthesia and analgesia in comparison with ropivacaine

Abstract

Background and objectives: Clonidine, buprenorphine, dexamethasone, and midazolam (C, B, D, M) have been used to prolong perineural local anesthesia in the absence of data on the influence of these adjuvants on local anesthetic-induced neurotoxicity. Therefore, the impact of these adjuvants on ropivacaine (R)-induced death of isolated sensory neurons was assessed.

Methods: The trypan blue exclusion assay was used to assess death of sensory neurons isolated from adult male Sprague-Dawley rats. Drugs were applied, alone or in combination, for 2 or 24 hrs at 37°C.

Results: Neuronal viability was halved by 24-hr exposure to R (2.5 mg/mL), far exceeding the neurotoxicity of C, B, D, or M (at 2-100 times estimated clinical concentrations). Plain M at twice the estimated clinical concentration produced a small but significant increase in neurotoxicity at 24 hrs. After 2-hr exposure, high concentrations of B, C, and M increased the neurotoxicity of R; the combination of R + M killed more than 90% of neurons. Estimated clinical concentrations of C + B (plus 66 μg/mL D) had no influence on (i) R-induced neurotoxicity, (ii) the increased neurotoxicity associated with the combination of R + M, or (iii) the neurotoxicity associated with estimated clinical concentrations of M. There was increased neurotoxicity with 133 μg/mL D combined with R + C + B.

Conclusions: Results with R reaffirm the need to identify ways to mitigate local anesthetic-induced neurotoxicity. While having no protective effect on R-induced neurotoxicity in vitro, future research with adjuvants should address if the C + B + D combination can enable reducing R concentrations needed to achieve equianalgesia (and/or provide equal or superior duration, in preclinical in vivo models).

Figure 1

Cell death as a function of treatment with ropivacaine (R) and high-concentration (see Table 1) perineural adjuvant single-drugs (C: clonidine, B: buprenorphine, D: dexamethasone, M: midazolam) without R, after 24 hr. Two concentrations of M were tested (33.3 and 66.6 µg/mL, M33 and M66, respectively). Based on ANOVA with the Holm-Sidak correction for multiple comparisons, M66, C, B, and R were cytotoxic to neurons at 24 hr, but M33 and D were not. *P≤0.012 (versus choline), †P<0.001 (versus choline and all other treatments), ‡P<0.001 for B being less neurotoxic than R, and P≤0.007 for B being more neurotoxic than all treatments other than R.

Figure 2

Cell death as a function of treatment and time, for ropivacaine (R) combinations with high-concentrations (Table 1) of single adjuvants clonidine (C), buprenorphine (B), dexamethasone (D), and midazolam (M) after 2 hr of drug exposure. Based on ANOVA with the Holm-Sidak correction for multiple comparisons, R-M and R-C were significantly more neurotoxic than choline (*P<0.001) and were more neurotoxic than R, R-D, and R-B (*P<0.01). R-B was neurotoxic compared to choline control (†P=0.046). Based on post hoc comparisons, C and M as single-adjuvants worsened R toxicity (each P<0.001), D and B did not. ‡P<0.01 for R-B being less cytotoxic than each of R-C and R-M).

Figure 3

Cell death as a function of treatment after 24 hr exposure to plain ropivacaine (R) and clonidine (C) – buprenorphine (B) – dexamethasone (D) - midazolam (M) adjuvant combinations (in estimated clinical concentrations, Table 1) without and with R. Concentrations of M are listed in the figure as 16.6 µg/mL (M17) and as 33.3 µg/mL (M33); concentrations of D are similarly listed as 66.6 µg/mL (D66) and as 133.3 µg/mL (D133). Of note, D66 and D133 are technically lower concentrations than those that have been reported clinically in single-injection blocks (see Methods text for details) Figure 3a shows all R-M combinations, with plain R as a reference for post hoc comparisons. Based on ANOVA with the Holm-Sidak correction for multiple comparisons, M17 and M33 combined with RPV were profoundly neurotoxic (independent of the presence of C-B-D); *P<0.001 for each R drug (plain or mixture) being more significantly neurotoxic than choline control, and †P<0.001 for plain R being less cytotoxic than all R combinations. Figure 3b shows the cytotoxicity outcomes after 24 hr exposure for the listed adjuvant combinations in “estimated clinical” concentrations (Table 1), without and with R 2.5 mg/mL (with no combinations of R and M). Based on ANOVA with the Holm-Sidak correction for multiple comparisons, C-B-D-M17 and C-B-D-M-33 were not cytotoxic, while all R (plain or in combination) mixtures were (*P<0.001). C-B-D66 did not worsen the cytotoxicity of plain R, but C-B-D133 did worsen the neurotoxicity of plain R (†P=0.045).