Restorative Neurology and Neuroscience xx (20xx) x–xx 1

DOI 10.3233/RNN-190987
IOS Press

1 Distinct neurotoxic effects of select

2 local anesthetics on facial nerve injury
and recovery

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4 Susanna C. Byrama,b,c,∗ , Samantha E. Bialekb,c , Vicki A. Husakb , Daniel Balcarcelc,1 , James Parkc,2 ,
5 Jacquelyn Dangc and Eileen M. Foeckingb,d
a Department of Anesthesiology, Loyola University Medical Center, Maywood, IL, (Byram – current), USA

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6

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b Research Service, Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines, IL, (Byram - current,
8 Bialek - current, Husak - current, Foecking - current), USA
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c Stritch School of Medicine, Loyola University Medical Center, Maywood, IL, (Byram - current, Balcarcel -

10 previous, Park – previous, Bialek – current, Dang - current), USA

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d Department of Otolaryngology—Head and Neck Surgery, Loyola University Medical Center, Maywood, IL,

12 (Foecking - current), USA

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13 Abstract.
14 Background: Local anesthetic toxicity has been well-documented to cause neuronal injury, death, and dysfunction, partic-
15 ularly in a susceptible nerve.
16 Objective: To determine whether select local anesthetics affect neuron survival and/or functional recovery of an injured
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17 nerve.
18 Methods: This report describes 6 separate experiments that test immediate or delayed application of local anesthetics in
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19 3 nerve injury models. Adult C57/black6 male mice underwent a facial nerve sham, transection, or crush injury. Local
20 anesthetic or saline was applied to the facial nerve at the time of injury (immediate) or 1 day after injury (delayed). Average
21 percent facial motoneuron (FMN) survival was evaluated four-weeks after injury. Facial nerve regeneration was estimated
22 by observing functional recovery of eye blink reflex and vibrissae movement after facial nerve crush injury.
23 Results: FMN survival after: transection + immediate treatment with ropivacaine (54.8%), bupivacaine (63.2%), or tetracaine
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24 (66.9%) was lower than saline (85.5%) and liposomal bupivacaine (85.0%); crush + immediate treatment with bupivacaine
25 (92.8%) was lower than saline (100.7%) and liposomal bupivacaine (99.3%); sham + delayed treatment with bupivacaine
26 (89.9%) was lower than saline (96.6%) and lidocaine (99.5%); transection + delayed treatment with bupivacaine (67.3%) was
27 lower than saline (78.4%) and liposomal bupivacaine (77.6%); crush + delayed treatment with bupivacaine (85.3%) was lower
than saline (97.9%) and lidocaine (96.0%). The average post-operative time for mice to fully recover after: crush + immediate
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28

29 treatment with bupivacaine (12.83 days) was longer than saline (11.08 days) and lidocaine (10.92 days); crush + delayed
30 treatment with bupivacaine (16.79 days) was longer than saline (12.73 days) and lidocaine (11.14 days).
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∗ Corresponding author: Susanna C. Byram, MD, PhD, Loy-

ola University Medical Center, 2160 S. First Ave. Maywood, IL,

60153, USA. Tel.: +1 708 216 3754; Fax: +1 708 216 8267; E-mail:
Susanna.Byram@lumc.edu.
1 Department of Pediatrics, Northwestern University Feinberg

School of Medicine, Ann & Robert H. Lurie Children’s Hospital

of Chicago, Chicago, IL (Balcarcel - current), USA.
2 Department of Anesthesia, Brown University/Rhode Island

Hospital, Providence, RI, (Park – current) , USA.

0922-6028/20/$35.00 © 2020 – IOS Press and the authors. All rights reserved
 2 S.C. Byram et al. / Local anesthetics on facial nerve injury

31 Conclusions: Our data demonstrate that some local anesthetics, but not all, exacerbate motoneuron death and delay functional
32 recovery after a peripheral nerve injury. These and future results may lead to clinical strategies that decrease the risk of neural
33 deficit following peripheral nerve blocks with local anesthetics.

Keywords: Neurotoxicity, motoneuron survival, peripheral nerve block, local anesthetic toxicity, peripheral nerve injury,
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facial nerve injury, liposomal bupivacaine, regional anesthesia
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31 1. Introduction well described facial nerve injury model to evalu- 74

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ate the neurotoxic potential of commonly used local 75

32 Local anesthetics have been used in the periopera- anesthetics. 76

33 tive period to alleviate pain for more than 100 years.

34 Local anesthetics exert their effects by reversibly
35 binding to voltage-gated sodium channels on neurons 2. Materials and methods 77

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36 to interrupt electrical impulse propagation (Nau &
37 Wang, 2004). However, nerve damage can occur from 2.1. Animals 78

38 peripheral nerve block procedures and lead to new

39 or worsened functional impairment for patients. One This manuscript adheres to the applicable EQUA- 79

mechanism of nerve damage is direct neurotoxicity

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40 TOR guidelines (ARRIVE guidelines). 245 adult 80

41 of local anesthetics, however the pathways involved male C57B/6 mice (7-8 weeks old; 22g–26 g; 81

42 are incompletely understood (Hogan, 2008; Lirk et N = 5–12 per group) were obtained from Jackson 82

43 al., 2008; Selander, 1993). The question arises, there- Laboratories (Bar Harbor, ME). All mice were 83

44 fore, whether nerves injured at the time of surgery housed and manipulated in accordance with institu- 84
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45 are affected by direct exposure to local anesthetics tional and National Institutes of Health guidelines 85

46 either pre-, intra-, or post-procedure (i.e. after clos- and approved by the Animal Care and Use Com- 86

47 ing a wound). Tracking the incidence and recovery mittee at Edward Hines Jr., VA Hospital (Hines, IL) 87

48 of perioperative nerve injury is challenging. Sub- to conduct the following experiments. Three sep- 88

49 jective sensory testing is fraught with uncertainties, arate experimental injury models were studied; 1) 89

and post-operative motor testing can be limited by facial nerve sham, 2) facial nerve transection, 3)
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50 90

51 apprehension, pain, dressings, patient expectations, facial nerve crush. Two separate local anesthetic 91

etc. Even with objective electromyography studies,

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52 application times were studied: 1) immediate local 92

53 it is difficult to determine if a post-operative nerve anesthetic treatment at the time of injury and 2) 93

54 deficit is related to a nerve block, iatrogenic surgi- delayed local anesthetic treatment, 1 day after the 94

55 cal injury, or another mechanism. Because of these initial injury. For each experiment in the immediate 95

56 confounding factors, the incidence rates of periph- treatment study, the mice were randomly divided into 96
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57 eral nerve dysfunction range widely after peripheral one of eight treatment groups: vehicle (saline), lido- 97

58 nerve block reported in the literature (Liguori, 2004; caine, 2, 3-chloroprocaine, mepivacaine, ropivacaine, 98

59 Liu et al., 2009). Early transient postoperative neuro- bupivacaine, tetracaine, or liposomal bupivacaine 99

60 logic symptoms are very common with some studies (Exparel; Pacira Pharmaceuticals, Parsippany, NJ). 100
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61 reporting up to 14% of patients complaining of For each experiment in the delayed treatment study, 101

62 dysesthesia, paresthesia, or mild pain in the immedi- the mice were randomly divided into one of four 102

63 ate perioperative period, however long-term clinical treatment groups: vehicle (saline), lidocaine, bupiva- 103

64 implications of these symptoms are unknown (Brull, caine, or liposomal bupivacaine. The local anesthetic 104
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65 McCartney, Chan, & El-Beheiry, 2007; Liguori, treatment groups were blinded to all investigators 105

66 2004; Liu et al., 2009). We hypothesize that the effects throughout the study until after data analysis was 106

67 of local anesthetic in the setting of nerve injury may completed. Animal groups were coded by one inves- 107

68 result in worsening facial nerve function in a mouse tigator and subsequently analyzed under “blind” 108

69 nerve injury model. The objective of this study, there- conditions by a second and third investigator, who 109

70 fore, was to determine if select local anesthetics can were unaware of the injury and treatment group 110

71 exacerbate neuron cell death and/or adversely delay divisions. 111

72 functional recovery when applied to an injured facial For all surgical procedures, mice were fully anes- 112

73 nerve. For proof of concept, we have utilized the thetized using 2% inhaled isoflurane and aseptic 113
 S.C. Byram et al. / Local anesthetics on facial nerve injury 3

114 technique followed. In all 3 injury models, an inci- based on the maximal suggested dose used clinically 163

115 sion was made behind the right ear and the right by weight. 164

116 facial nerve was exposed at its exit from the stylo-
117 mastoid foramen. For the sham surgery, no nerve 2.3. Data collection 165

118 injury was performed. For the transection injury,

119 the right facial nerve was completely transected at 2.3.1. Facial motoneuron survival 166

120 the stylomastoid foramen using iridectomy scissors. Four weeks post-operatively all animals were 167

121 The proximal and distal nerve stumps were manu- euthanized via isoflurane overdose followed rapidly 168

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122 ally displaced to prevent reconnection. Animals in by decapitation and the brains were removed and 169

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123 the sham and transection groups were observed bi- promptly frozen in a solution of butyl-bromide 170

124 weekly to confirm presence or absence of facial and 2-methylbutane cooled with dry ice to below 171

125 nerve function by evaluating eye blink or vibrissae –40◦ C. Twenty-five ␮m thick sections were col- 172

126 movement. For the crush injury, the facial nerve was lected throughout the facial nucleus, fixed in 4% 173

127 crushed twice for 30 seconds each, using Dumont paraformaldehyde and stained with thionin to reveal 174

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128 #5/45 forceps held at different orientations to the motoneuron cell bodies (Fig. 1). One investigator 175

129 nerve. All wounds were closed with 5-0 prolene coded all slides and animal groups and a second 176

130 monofilament suture. In the delayed local anesthetic and third investigator determined surviving FMN 177

131 treatment groups, mice underwent a second surgery to counts in each section using light microscopy. The 178

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132 re-expose the intact (sham) or injured (transection or abducens nuclei and internal genu of the facial nerves 179

133 crush) nerves one day after injury, for local anesthetic were used to precisely match the sections of the 180

134 application. left (uninjured) and right (injured) sides. Using 100x 181

magnification, surviving FMNs were easily identified 182

135 2.2. Local anesthetic treatment by morphologic recognizability as nucleated multi- 183

polar cells with somatic cell size ∼25–40 ␮m and

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136 2.2.1. Immediate local anesthetic application only motoneurons containing a clear nucleus (see 185

137 At the time of facial nerve injury, one of inset Fig. 1A) were counted and compared on a mini- 186

138 seven commonly used local anesthetics (2% lido- mum of 15 location-matched sections per animal. The 187

139 caine [7 mg/kg], 3% 2, 3-chloroprocaine [15 mg/kg], percentage change between the uninjured and injured 188

1% mepivacaine [7 mg/kg], 0.25% ropivacaine sides was calculated and the average percent survival
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140 189

141 [5 mg/kg], 0.25% bupivacaine [5 mg/kg], 1% tetra- recorded for each animal. 190
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142 caine [2 mg/kg], or 1.3% liposomal bupivacaine

143 [5 mg/kg]), or vehicle (0.9% normal saline), was 2.3.2. Functional recovery assessments 191

144 applied to the facial nerve in a total volume of Facial nerve crush injury results in loss of eye 192

145 50␮l via impregnated Gelfoam (Pfizer; New York, blink reflex, vibrissae movement and abnormal vib- 193

146 New York). Gelfoam was chosen in an attempt to rissae orientation with fibers flattened in a posterior 194
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147 sequester the local anesthetic to the site of injury direction against the head (Kujawa, Kinderman, & 195

148 and potentially avoid run-off into the surrounding Jones, 1989). Recovery post-crush injury is consid- 196

149 tissue. The total dose for each local anesthetic was ered complete when all three parameters are equal 197

150 based on the maximal suggested dose used clinically in magnitude and orientation relative to the unin- 198
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151 by weight. jured (control) side. Animals in the nerve crush injury 199

study were observed twice daily by 2 observers that 200

152 2.2.2. Delayed local anesthetic application were blinded to the treatment group. The eye blink 201

153 One day after facial nerve injury, one of three reflex on the injured side was observed in compar- 202
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154 commonly used local anesthetics (2% lidocaine ison to the uninjured side, by carefully exhaling a 203

155 [7 mg/kg], 0.25% bupivacaine [5 mg/kg], or 1.3% gentle puff of air directed at the animal’s corneas. 204

156 liposomal bupivacaine [5 mg/kg]), or vehicle (0.9% Eye blink was assessed utilizing a 4-point scale; 205

157 normal saline), was applied directly to the facial nerve 1-no blink, 2-slight blinking movement, 3-robust 206

158 injury site via syringe in a total volume of 50␮l. Of eye blink but not matching the control side, and 4- 207

159 note, Gelfoam was not used in these studies to avoid full blink, matching the control side. Evidence of 208

160 its potential to confound the pharmacokinetics and bulbar retraction with passive eyelid closing was 209

161 pharmacodynamics of treatment. See Discussion for carefully observed to avoid over-scoring. Vibrissae 210

162 details. The total dose for each local anesthetic was movement was assessed utilizing a 4-point scale; 1-no 211
 4 S.C. Byram et al. / Local anesthetics on facial nerve injury

scale; 1-posterior or “flattened” against the face, 2- 216

away from face but not matching the control side, 217

and 3-fully anterior, matching the control side. Care- 218

ful consideration of contralateral intact vibrissae pad 219

movement was taken to ensure accurate assessments. 220

In the event whiskers were groomed off an animal 221

by a cage mate and observers were unable to make 222

reasonable behavioral assessments, the animals were 223

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removed from the behavioral analysis. 224

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2.4. Statistical analysis 225

For FMN survival data, in accordance with our pre- 226

viously published work, the Abercrombie correction 227

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factor (N = n×T/T+D), where N is the actual num- 228

ber of cells, n is the number of nuclear profiles, T is 229

the section thickness (25 ␮m), and D is the average 230

diameter of nuclei (5 ␮m) (Byram, Byram, Miller, & 231

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Fargo, 2017; Coggeshall, 1992), was used to com- 232

pensate for double counting in adjacent sections. The 233

percent change in the number of FMNs between the 234

uninjured and injured facial motor nuclei was deter- 235

mined for each animal and then the average percent 236
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survival and standard error of the mean (SEM) deter- 237

mined for each experimental group. 238

For animals receiving a crush injury, the aver- 239

age time in post-operative days until the onset of 240

complete recovery of vibrissae orientation, eyeblink 241

reflex, and vibrissae movement, as well as the onset

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242

of complete recovery of all three of these facial nerve 243

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functions, was determined for each animal. The aver- 244

age number of days and SEM was then determined 245

for each experimental group of the crush injured 246

animals. 247

A one-way analysis of variance (ANOVA) was 248

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conducted to compare the effects of select local 249

anesthetics on FMN survival and time to complete 250

recovery. Post-hoc comparisons using the Tukey’s 251

Fig. 1. Thionin-stained facial motoneurons. Representative pho-

multiple comparison test was done to identify local 252
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tomicrographs of thionin-stained facial motoneurons in the anesthetics that exert significant effects on the out- 253

uninjured (no transection) and injured (transection) facial nuclei come measures (GraphPad Prism 8). 254
of mice treated with saline (A and B), lidocaine (C and D), 2,3-
chloroprocaine (E and F), mepivacaine (G and H), ropivacaine (I
and J), bupivacaine (K and L), tetracaine (M and N), and liposomal
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bupivacaine (O and P) at the time of injury (immediate treatment). 3. Results 255

Scale bar - 50 ␮m. Inset of 1a is a magnified example motoneuron,

demonstrating characteristic morphology and presence of distinct 3.1. FMN Survival 256
nucleus used for cell counting.

3.1.1. Immediate local anesthetic application 257

212 movement, 2-slight movement, 3-robust movement To determine if commonly used local anesthetics 258

213 but not equal to the control side, and 4-full move- affect neuronal survival after a peripheral nerve sham, 259

214 ment matching and coordinated with the control side. transection, or crush injury, we applied saline, lido- 260

215 Vibrissae orientation was assessed utilizing a 3-point caine, 2, 3-chloroprocaine, mepivacaine, ropivacaine, 261
 S.C. Byram et al. / Local anesthetics on facial nerve injury 5

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Fig. 2. Average percent facial motoneuron survival after immediate local anesthetic treatment. Bar graph demonstrating average percentage

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of facial motoneuron survival at 4 weeks after facial nerve injury and immediate treatment with saline, lidocaine, 2,3-chloroprocaine,
mepivacaine, ropivacaine, bupivacaine, tetracaine, and liposomal bupivacaine. A – Sham injury, B – Transection injury, C – Crush injury.
* denotes statistical difference compared to ropivacaine (P < 0.05). # denotes statistical difference compared to bupivacaine (P < 0.05). 
denotes statistical difference compared to tetracaine (P < 0.05).

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262 bupivacaine, tetracaine, or liposomal bupivacaine to decreased compared to saline and liposomal bupi- 295

263 the facial nerve immediately at the time of injury. vacaine. And the average percent FMN survival 296

264 Four weeks post sham injury and immediate after transection in mice immediately treated with 297

265 local anesthetic treatment, the average percent tetracaine (66.9% ± 4.3, P < 0.05; n = 8) was signif- 298
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266 FMN survival in mice was determined. A one- icantly decreased compared to saline and liposomal 299

267 way ANOVA conducted to compare the effect of bupivacaine. 300

268 7 local anesthetics on FMN survival after facial Four weeks post crush injury and immediate local 301

269 nerve sham injury revealed no statistical differ- anesthetic treatment, the average percent FMN sur- 302

270 ence for treatment with saline (97.8% ± 1.9; n = 6), vival in mice was determined. A one-way ANOVA 303

lidocaine (95.8% ± 2.0; n = 6), 2, 3-chloroprocaine

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271 conducted to compare the effect of 7 local anes- 304

272 (98.7% ± 0.9; n = 5), mepivacaine (98.1% ± 1.2; thetics on FMN survival after facial nerve crush 305

n = 6), ropivacaine (94.5% ± 1.6; n = 6), bupivacaine

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273 revealed a statistically significant treatment effect [F 306

274 (92.4% ± 2.9; n = 6), tetracaine (94.8% ± 1.8, n = 6), (7, 53) = 2.633, P = 0.02] (Fig. 2C). Post-hoc compar- 307

275 or liposomal bupivacaine (94.9% ± 1.7; n = 6) [F (7, isons revealed that the average percent FMN survival 308

276 39) = 1.304, P = 0.27] (Fig. 2A). after crush in mice immediately treated with bupiva- 309

277 Four weeks post transection injury and immedi- caine (92.8% ± 0.9, P < 0.05; n = 9) was significantly 310
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278 ate local anesthetic treatment, the average percent decreased compared to saline (100.7% ± 1.8; n = 6) 311

279 FMN survival in mice was determined. A one- and liposomal bupivacaine (99.3% ± 1.0; n = 8). 312

280 way ANOVA conducted to compare the effect of 7 However, there were no statistically significant dif- 313

281 local anesthetics on FMN survival after facial nerve ferences between lidocaine (96.9% ± 1.3; n = 12), 314

2, 3-chloroprocaine (96.2% ± 1.1; n = 7), mepiva-

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282 transection revealed a statistically significant treat- 315

283 ment effect [F (7, 51) = 8.039, P < 0.0001] (Fig. 1 caine (95.3% ± 2.6; n = 6), ropivacaine (97.4% ± 1.1; 316

284 and Fig. 2B). Post-hoc comparisons revealed that n = 8), or tetracaine (97.8% ± 2.5; n = 5). 317

285 the average percent FMN survival after transec-

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286 tion in mice immediately treated with ropivacaine

287 (54.8% ± 4.7, P < 0.05; n = 7) was significantly 3.3.2. Delayed local anesthetic application 318

288 decreased compared to saline (85.5% ± 1.5; n = 6), To determine if delayed local anesthetic admin- 319

289 lidocaine (72.9% ± 5.2; n = 6), 2, 3-chloroprocaine istration affects neuronal survival after a peripheral 320

290 (75.6% ± 2.4; n = 8), mepivacaine (77.3% ± 4.6; nerve sham, transection, or sham injury, we applied 321

291 n = 8), and liposomal bupivacaine (85.0% ± 2.9; saline, lidocaine, bupivacaine, or liposomal bupiva- 322

292 n = 8). The average percent FMN survival after caine to the facial nerve 1 day after facial nerve 323

293 transection in mice immediately treated with bupiva- injury. Of note, based on clinical trends and the prior 324

294 caine (63.2% ± 2.3, P < 0.05; n = 8) was significantly experiments, fewer local anesthetics were evaluated 325
 6 S.C. Byram et al. / Local anesthetics on facial nerve injury

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Fig. 3. Average percent facial motoneuron survival after delayed local anesthetic treatment. Bar graph demonstrating average percentage
of facial motoneuron survival at 4 weeks after facial nerve injury and delayed treatment with saline, lidocaine, bupivacaine, and liposomal
bupivacaine. A – Sham injury, B – Transection injury, C – Crush injury. # denotes statistical difference compared to bupivacaine (P < 0.05).

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326 in the delayed-treatment experiments. Furthermore, way ANOVA conducted to compare the effect of 363

327 to avoid the potential confounding treatment effects 3 local anesthetics on FMN survival after facial 364

328 of Gelfoam, local anesthetic was administered as a nerve crush injury revealed a statistically significant 365

solution via syringe directly to the nerve. treatment effect [F (3, 21) = 6.068, P = 0.003]. Post-

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329 366

330 Four weeks post sham injury and delayed local hoc comparisons revealed that the average percent 367

331 anesthetic treatment, the average percent FMN FMN survival after crush injury in mice delay- 368

332 survival in mice was determined (Fig. 3A). A one- treated with bupivacaine (85.3% ± 2.2, P < 0.05; 369

333 way ANOVA conducted to compare the effect of n = 5) was significantly decreased compared to saline 370

(97.9% ± 2.2; n = 7) and lidocaine (96.0% ± 1.4;

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334 3 local anesthetics on FMN survival after facial 371

335 nerve sham injury revealed a statistically significant n = 8). However there were no statistically signifi- 372

336 treatment effect [F (3, 17) = 6.939, P = 0.003]. Post- cant differences compared to liposomal bupivacaine 373

337 hoc comparisons revealed that the average percent (92.8% ± 3.2; n = 5). 374

338 FMN survival after sham-injury in mice delay-

treated with bupivacaine (89.9% ± 1.8, P < 0.05; 3.2. Functional recovery
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339 375

340 n = 6), was significantly decreased compared to saline

(96.6% ± 2.0; n = 5) and lidocaine (99.5% ± 1.1;
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341 3.2.1. Immediate local anesthetic application 376

342 n = 5). However, there were no statistically signifi- To determine if commonly used local anesthet- 377

343 cant differences comparted to liposomal bupivacaine ics affect functional recovery after a peripheral 378

344 (96.0% ± 1.2; n = 5). nerve crush injury, we applied saline, lidocaine, 2, 379

345 Four weeks post transection and delayed local 3-chloroprocaine, mepivacaine, ropivacaine, bupiva- 380
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346 anesthetic treatment, the average percent FMN sur- caine, tetracaine, or liposomal bupivacaine to the 381

347 vival in mice was determined (Fig. 3B). A one-way facial nerve immediately at the time of injury. 382

348 ANOVA conducted to compare the effect of 3 The average post-operative time for mice to fully 383

349 local anesthetics on FMN survival after facial nerve recover, defined as full recovery of all three func- 384
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350 transection injury revealed a statistically signifi- tional parameters (eye blink, vibrissea orientation, 385

351 cant treatment effect [F (3, 20) = 6.53, P = 0.003]. vibrissae movement), was determined (Fig. 4A). A 386

352 Post-hoc comparisons using revealed that the aver- one-way ANOVA conducted to compare the effect 387

353 age percent FMN survival after transection in of 7 local anesthetics on functional recovery after 388
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354 mice delay-treated with bupivacaine (67.3% ± 1.2, facial nerve crush revealed a statistically signifi- 389

355 P < 0.05; n = 7) was significantly decreased compared cant treatment effect [F (7, 42) = 3.008, P = 0.01]. 390

356 to saline (78.4% ± 3.1; n = 6) and liposomal bupiva- Post-hoc comparisons revealed that the average post- 391

357 caine (77.6% ± 1.3; n = 6). However, there were no operative time for mice to fully recover after crush 392

358 statistically significant differences compared to lido- injury and immediate treatment with bupivacaine 393

359 caine (71.6% ± 2.6; n = 5). (12.83 ± 0.44 days, P < 0.05; n = 6) was significantly 394

360 Four weeks post crush injury and delayed local delayed compared to saline (11.08 ± 0.33 days; n = 6) 395

361 anesthetic treatment, the average percent FMN and lidocaine (10.92 ± 0.20 days; n = 6). However 396

362 survival in mice was determined (Fig. 3C). A one- there were no statistically significant differences 397
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Fig. 4. Functional recovery after crush injury and local anesthetic treatment. Bar graph demonstrating average post-operative time in days
until animals in crush injury groups achieved complete recovery of vibrissae orientation, vibrissae movement, and eyeblink. A – Immediate
local anesthetic treatment, B – Delayed local anesthetic treatment. # denotes statistical difference compared to bupivacaine (P < 0.05).

compared with 2, 3-chloroprocaine (11.67 ± 0.53 increased risk of new or worsening nerve damage.

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398 429

399 days; n = 6), mepivacaine (11.17 ± 0.33 days; n = 6), Similarly, the questions arise as to whether all local 430

400 ropivacaine (12.08 ± 0.45 days; n = 6), tetracaine anesthetics display a similar neurotoxic profile in 431

401 (11.25 ± 0.36 days; n = 6), or liposomal bupivacaine all patients. At present, analgesia can be adapted 432

402 (12.06 ± 0.29 days; n = 8). to a clinical scenario with the availability of sev- 433
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403 To determine if delayed treatment with commonly eral local anesthetics with varying properties, such 434

404 used local anesthetics affect functional recovery after as speed of onset, potency, and/or duration of action. 435

405 a peripheral nerve crush injury, we applied saline, With the ever-expanding use of regional anesthesia 436

406 lidocaine, bupivacaine, or liposomal bupivacaine to and push for improved pain control in all patients, 437

407 the facial nerve 1-day after injury. The average local anesthetics are frequently applied to peripheral 438

post-operative time for mice to fully recover was nerves that may already be diseased (i.e.: diabetic
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408 439

409 determined (Fig. 4B). A one-way ANOVA conducted neuropathy), injured, or at risk of injury during 440
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410 to compare the effect of delayed treatment with 3 surgery (i.e. mechanical, stretch, etc.). It has been 441

411 local anesthetics on functional recovery after facial well documented in vitro that all local anesthetics 442

412 nerve crush revealed a statistically significant treat- are toxic to neurons and their support cells as a 443

413 ment effect [F (3, 27) = 4.743, P = 0.008]. Post-hoc function of potency, concentration, dose, and dura- 444

414 comparisons revealed that the average post-operative tion of exposure (Boselli et al., 2003; Epstein-Barash 445
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415 time for mice to fully recover after crush injury and et al., 2009; Kalichman, Powell, & Myers, 1989; 446

416 delayed treatment with bupivacaine (16.79 ± 2.10 Lambert, Lambert, & Strichartz, 1994; Ohtake et 447

417 days, P < 0.05; n = 7) was significantly delayed com- al., 2000; Perez-Castro et al., 2009; Radwan, Saito, 448

418 pared to saline (12.73 ± 0.37 days; n = 11) and & Goto, 2002; Sakura, Bollen, Ciriales, & Drasner, 449

lidocaine (11.14 ± 0.66 days; n = 7). However there

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419 1995; Werdehausen et al., 2009; Yang, Abrahams, 450

420 were no statistically significant differences com- Hurn, Grafe, & Kirsch, 2011). Despite their neu- 451

421 pared with liposomal bupivacaine (12.92 ± 0.65 rotoxic potential, current opinion holds that local 452

422 days; n = 6). anesthetic concentrations at doses used clinically 453

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are likely safe for all patients, despite a paucity 454

of clinical studies comparing dose or concentration 455

423 4. Discussion between patients. However, the American Society of 456

Regional Anesthesia has suggested careful consider- 457

424 The benefits of providing local anesthetic and ation be taken in patients with pre-existing neurologic 458

425 nerve blocks for patients undergoing surgery have deficits for regional anesthesia due to the increased 459

426 been well established however it is less clear whether risk of exacerbating their underlying nerve dys- 460

427 individuals with preexisting neurologic deficits also function, and suggests limiting concentration, dose 461

428 benefit from regional anesthesia or are they at and avoidance of adjuvants in at-risk patients (Neal 462
 8 S.C. Byram et al. / Local anesthetics on facial nerve injury

463 et al., 2015). For the present study, we employed will investigate differences in local anesthetic toxi- 515

464 the well-described mouse facial nerve injury model city based on application timing from immediate to 516

465 to evaluate the neurotoxic potential of commonly variable lengths of delay. 517

466 used local anesthetics. We hypothesized that previ- After immediate local anesthetic application at the 518

467 ously injured neurons will be more susceptible to time of facial nerve transection, ropivacaine, bupiva- 519

468 the toxicity of certain commonly used local anes- caine, and tetracaine caused increased FMN death 520

469 thetics and will result in increased motoneuron cell compared to saline. Similarly, after delayed local 521

470 death and possibly worsened or delayed functional anesthetic application after facial nerve transection, 522

f
471 recovery. bupivacaine caused increased FMN death compared 523

roo
472 Our present data demonstrate differential toxic- to saline (ropivacaine and tetracaine were not stud- 524

473 ity between local anesthetics on an injured nerve ied in the delayed experiments). Following a facial 525

474 despite a common mechanism of action between all nerve crush injury, only bupivacaine caused increased 526

475 local anesthetics. In a previously published study, FMN death and delayed functional recovery when 527

476 we demonstrated that bupivacaine, but not lido- applied immediately or delayed as compared to 528

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477 caine, exacerbated FMN death after a facial nerve saline. The short and intermediate acting local anes- 529

478 transection (Byram et al., 2017). Others have also thetics (lidocaine, 2, 3-chloroprocaine, mepivacaine) 530

479 demonstrated the neurotoxicity of bupivacaine in did not exacerbate FMN death nor delay functional 531

480 vitro (Lirk et al., 2008; Werdehausen et al., 2009; recovery in any injury model when applied imme- 532

tho
481 Yamashita et al., 2003; Yang et al., 2011). We pro- diately or delayed. Of note, our prior publication 533

482 posed that the preferential toxicity of bupivacaine reports FMN survival of 35% after facial nerve tran- 534

483 over lidocaine was related to the longer duration of section and immediate treatment with bupivacaine, 535

484 action (Byram et al., 2017). Therefore, in the present while the present study reports FMN survival of 536

485 study, we expanded our investigation to evaluate 7 63% (Byram et al., 2017). There are two major dif- 537
Au
486 local anesthetics with varying pharmacologic char- ferences between these studies: 1) Our prior study 538

487 acteristics (including variable durations of action) in used a much higher concentration of bupivacaine 539

488 3 different injury models (sham, transection, crush). (0.75%) compared to the present study (0.25%) and 540

489 Furthermore, since the application of a local anes- 2) our prior study did not use Gelfoam for application. 541

490 thetic immediately/simultaneously at the time of a We suspect the higher concentration of bupivacaine 542

nerve injury in clinical practice is likely a rare occur- and/or the application technique of infiltration ver-
d

491 543

492 rence and may expose the intracellular environment sus Gelfoam, may account for the difference in FMN 544
cte

493 to toxic levels of local anesthetic while the axolemma survival between studies. (See discussion on Gelfoam 545

494 is open, we repeated our experiments for select local below). 546

495 anesthetics using a delayed application model (1-day) Unexpectedly, treatment with bupivacaine 547

496 to allow the axolemma to seal and neuron survival increased FMN death compared to liposomal bupi- 548

497 and regenerative processes to initiate prior to local vacaine after transection injury in both immediate 549
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498 anesthetic exposure. and delayed application studies, as well as after crush 550

499 Similar to our previous findings, bupivacaine injury and immediate application. 1.3% liposomal 551

500 increased FMN cell death in 5 of the 6 experiments bupivacaine is a higher concentration bupivacaine 552

501 (immediate-transection, immediate-crush, delayed- formulation (compared to 0.25% free bupivacaine) 553
co

502 sham, delayed-transection, delayed-crush) when and is the longest-acting local anesthetic tested, 554

503 compared to a saline control. And while bupi- however it did not exacerbate FMN death nor delay 555

504 vacaine did cause the most FMN death in the functional recovery in any injury model tested. Thus, 556

505 immediate-sham experiment, its effects did not reach the idea that bupivacaine itself, total concentration, 557
Un

506 statistical significance. Unexpectedly, delayed bupi- dose, or duration of exposure are responsible for 558

507 vacaine application after a sham injury increased the neurotoxic effects described is not entirely true 559

508 FMN death compared to saline, suggesting toxicity for liposomal bupivacaine. Others have similarly 560

509 of bupivacaine even in the setting of an uninjured demonstrated the lack of nerve toxicity with lipo- 561

510 nerve. It is unknown if the difference between imme- somal bupivacaine as compared to free bupivacaine 562

511 diate and delayed experiments is due to application (Damjanovska et al., 2015; McAlvin et al., 2014). 563

512 method (Gelfoam vs infiltration), timing of appli- Perhaps the slow release of bupivacaine from a 564

513 cation (immediate vs delayed), or another reason. liposomal formulation exposes nerves to lower 565

514 (See discussion on Gelfoam below). Future studies concentrations and doses in a given time, resulting in 566
 S.C. Byram et al. / Local anesthetics on facial nerve injury 9

567 less neurotoxicity. This finding is noteworthy, needs 2012). However, the facial nerve injury model is 619

568 further study, and may have important implications a simple and reproducible peripheral nerve injury 620

569 in clinical practice. model that has been extensively studied for decades to 621

570 The mechanisms responsible for local anesthetic evaluate mechanisms related to nerve injury, survival, 622

571 neurotoxicity are not entirely understood and will and regeneration in addition to the evaluation of neu- 623

572 be the focus of future studies. Local anesthetics rotoxins and neurotrophic factors (Moran & Graeber, 624

573 not only target voltage-gated sodium channels, but 2004). Second, to ensure an effective double-crush 625

574 also interact with a variety of other receptors which nerve injury model, we compared the neurotoxicity of 626

f
575 may mediate their neurotoxic effects (Lirk et al., local anesthetics after a complete nerve transection, 627

roo
576 2014). For example, in vitro models of local anes- which is an admittedly uncommon clinical scenario. 628

577 thetic induced neurotoxicity have described effects Furthermore, the clinical or behavioral significance 629

578 on caspase-, PI3k-, and MAPK-pathways (Haller et of neuron death after local anesthetic administration 630

579 al., 2006; Ma et al., 2010; Verlinde et al., 2016; is unknown. Therefore, we also utilized a more com- 631

580 Werdehausen et al., 2007). However, these stud- monly encountered nerve crush injury. The facial 632

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581 ies do not evaluate the effects of local anesthetic nerve crush model is useful for determining both 633

582 application to an axon, but rather to a cell body in neuron death and to evaluate axonal regeneration 634

583 culture. Here, we have combined the application of by observing the recovery of facial motor func- 635

584 local anesthetics to a peripheral nerve injury model, tions. Here, we utilized a subjective observational 636

tho
585 and therefore separate mechanisms of neurotoxic- method with incremental scales to detect changes in 637

586 ity may be responsible for the findings described. functional recovery of vibrissae orientation, move- 638

587 Under usual conditions, peripheral nerve transec- ment, and eye blink reflex. We endeavored to reduce 639

588 tion results in increased expression of injury and observer bias by using 2 separate observers, and care- 640

589 regeneration associated genes in the cell body (Al- ful consideration of confounding influences such as 641
Au
590 Majed, Brushart, & Gordon, 2000; Kiryu-Seo & contralateral intact vibrissae pad movement and bul- 642

591 Kiyama, 2011). Interestingly, several studies have bar retraction with passive eyelid closing were taken. 643

592 demonstrated an increase in injury and regeneration This methodology does not provide a detailed or 644

593 associated gene expression induced by application quantitative biometric analysis of motor recovery as 645

594 of brief electrical stimulation proximal to axo- described by others, which will be the goal for future 646

tomy, that is abolished by blocking action potentials studies (Guntinas-Lichius et al., 2001). Despite these
d

595 647

596 with tetrodotoxin (Al-Majed, Brushart, et al., 2000; drawbacks, the present study does demonstrate that 648
cte

597 Al-Majed, Neumann, Brushart, & Gordon, 2000; after a nerve crush injury, bupivacaine causes facial 649

598 Geremia, Gordon, Brushart, Al-Majed, & Verge, neuron death as well as delay in functional recovery. 650

599 2007). Thus, one possible mechanism of local anes- Clinically, local anesthetics are applied via injec- 651

600 thetic toxicity in the setting of nerve injury may be tion into the perineuronal space which is typically 652

601 that blocking nerve transmission after injury impairs bounded by fascial planes. In our surgical model, 653
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602 propagation of action potentials to the cell body and the fascia has been dissected away to perform a 654

603 thus negatively affects the usual cell body response to nerve injury. To avoid local anesthetic spilling out 655

604 injury. However, given that all local anesthetics tested of the intended area we initially used local anesthetic 656

605 are known to block action potentials, it is intrigu- impregnated Gelfoam for the immediate-application 657
co

606 ing that some, but not all local anesthetics resulted experiments. Gelfoam is a water-insoluble, porous 658

607 in decreased FMN survival and delayed functional sponge made from purified porcine skin and gelatin 659

608 recovery in our study. Thus, another mechanism is that can absorb and hold many times its weight of 660

609 likely responsible. Future studies will evaluate pos- fluid (Pfizer, 2019). However, after recognizing that 661
Un

610 sible mechanisms causing variable neurotoxicity of bupivacaine and liposomal bupivacaine had differen- 662

611 local anesthetics. tial neurotoxicity, we appreciated that the delivery 663

612 Our studies have some important limitations. First, method, such as using liposomes or Gelfoam could 664

613 in clinical practice, the facial nerve is not com- skew our results. Sequestration of local anesthetic 665

614 monly a target for nerve blocks, however branches into the Gelfoam may affect the pharmacodynamics 666

615 of the facial nerve can be incidentally affected by and/or pharmacokinetics of the local anesthetics. For 667

616 local anesthetics during local infiltration for dental, example, Gelfoam sequestration may slow or delay 668

617 facial reconstructive, or otolaryngology procedures release of local anesthetic and change the concentra- 669

618 (Tzermpos, Cocos, Kleftogiannis, Zarakas, & Iatrou, tion or total dose interacting with the nerve at a given 670
 10 S.C. Byram et al. / Local anesthetics on facial nerve injury

671 time, and thus inadvertently decrease the significance approval of the manuscript for publication. The con- 714

672 of our findings. Additionally, Gelfoam may affect the tents of this manuscript do not represent the views of 715

673 inflammatory state at the injury site, and the differ- the U.S. Department of Veterans Affairs or the United 716

674 ential neurotoxicity reported here may be related to States Government. 717

675 the differences each of the drugs has on inflamma-

676 tion or inflammation has on the action of the drug.
677 Therefore, since Gelfoam is not used clinically dur- Access to data and data analysis 718
678 ing peripheral nerve blocks, we chose to remove it

f
679 from our follow-up studies. Ultimately, despite the Drs. Byram and Foecking had full access to all

roo
719
680 potentially confounding limitation of using Gelfoam the data in the study and take responsibility for the 720
681 in our immediate application studies, bupivacaine integrity of the data and the accuracy of the data 721
682 increased FMN death and delayed functional recov- analysis 722
683 ery after transection and/or crush injuries, validating
684 the negative effects of bupivacaine.

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685 There are ongoing questions regarding the effects
686 of local anesthetics and regional techniques on References 723

687 injured or diseased peripheral nerves, and there are Al-Majed, A.A., Brushart, T.M., & Gordon, T. (2000). Electrical 724
688 limited recommendations for these patients. Our cur- stimulation accelerates and increases expression of BDNF 725

tho
689 rent study has identified select local anesthetics to and trkB mRNA in regenerating rat femoral motoneurons. 726

690 help focus forthcoming studies. We hope that an European Journal of Neuroscience, 12(12), 4381-4390. 727

691 improved understanding of the effects of local anes- Al-Majed, A.A., Neumann, C.M., Brushart, T.M., & Gordon, T. 728
(2000). Brief electrical stimulation promotes the speed and 729
692 thetics on injured peripheral nerves may allow us accuracy of motor axonal regeneration. Journal of Neuro- 730
693 to prevent and/or minimize neural deficits following science, 20(7), 2602-2608. 731
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694 peripheral nerve blocks in at-risk patients. Boselli, E., Duflo, F., Debon, R., Allaouchiche, B., Chassard, 732
D., Thomas, L., & Portoukalian, J. (2003). The induction 733
of apoptosis by local anesthetics: a comparison between 734

695 Disclosures lidocaine and ropivacaine. Anesthesia and Analgesia, 96(3), 735
755-756. 736

Brull, R., McCartney, C.J., Chan, V.W., & El-Beheiry, H. (2007). 737
Dr. Byram has received payment for consultation
d

696
Neurological complications after regional anesthesia: con- 738
697 services from Pacira Pharmaceuticals, and ACI Clin- temporary estimates of risk. Anesthesia and Analgesia, 739
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698 ical. These activities were unrelated to the research 104(4), 965-974. doi:10.1213/01.ane.0000258740.17193.ec 740

699 activities described here. Byram, S.C., Byram, S.W., Miller, N.M., & Fargo, K.N. (2017). 741
Bupivacaine increases the rate of motoneuron death following 742
peripheral nerve injury. Restorative Neurology and Neuro- 743
science, 35(1), 129-135. doi:10.3233/RNN-160692 744
700 Funding/support
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Coggeshall, R.E. (1992). A consideration of neural counting meth- 745

ods. Trends in Neurosciences, 15(1), 9-13. 746
701 This work was supported in part (immediate
Damjanovska, M., Cvetko, E., Hadzic, A., Seliskar, A., Plavec, 747
702 local anesthetic administration) by The Depart- T., Mis, K.,... Stopar Pintaric, T. (2015). Neurotoxicity of 748
703 ment of Anesthesia at Loyola University Medical perineural vs intraneural-extrafascicular injection of liposo- 749
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704 Center (Maywood, Illinois USA). This work was mal bupivacaine in the porcine model of sciatic nerve block. 750

705 also supported in part (delayed local anesthetic Anaesthesia, 70(12), 1418-1426. doi:10.1111/anae.13189 751

706 administration) by Small Projects in Rehabilita- Epstein-Barash, H., Shichor, I., Kwon, A.H., Hall, S., Lawlor, 752
M.W., Langer, R., & Kohane, D.S. (2009). Prolonged dura- 753
707 tion Research, Award # RX002228-01A1, from the
Un

tion local anesthesia with minimal toxicity. Proceedings of the 754

708 United States Department of Veterans Affairs Reha- National Academy of Sciences of the United States of America, 755
709 bilitation Research & Development Service. 106(17), 7125-7130. doi:10.1073/pnas.0900598106 756

Geremia, N.M., Gordon, T., Brushart, T.M., Al-Majed, A.A., 757

& Verge, V.M. (2007). Electrical stimulation promotes 758

710 Role of the funder/sponsor sensory neuron regeneration and growth-associated gene 759
expression. Experimental Neurology, 205(2), 347-359. 760
doi:10.1016/j.expneurol.2007.01.040 761
711 The sponsors had no role in the design and con-
Guntinas-Lichius, O., Angelov, D.N., Tomov, T.L., Dramiga, J., 762
712 duct of the study, collection, management, analysis, Neiss, W.F., & Wewetzer, K. (2001). Transplantation of olfac- 763
713 and interpretation of the data, preparation, review, or tory ensheathing cells stimulates the collateral sprouting from 764
 S.C. Byram et al. / Local anesthetics on facial nerve injury 11

765 axotomized adult rat facial motoneurons. Experimental Neu- Nau, C., & Wang, G.K. (2004). Interactions of local anesthet- 823
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810 doi:10.1213/ane.0b013e3181a3272c induction by different local anaesthetics in a neuroblastoma 868

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819 35(15), 4557-4564. doi:10.1016/j.biomaterials.2014.02.015 Kirsch, J. R. (2011). Local anesthetic Schwann 877
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