Introduction
============

Adequate pain management has been shown to improve the rate and quality of patient recovery following surgery ([@B48]). Peripheral nerve blocks (PNBs) using local anesthetic (LA) are superior to opioid analgesia which have a high rate of undesirable systemic effects ([@B46]; [@B1]). Voltage-gated Na+ channel (VGSC) blockers acting as LA (e.g. bupivacaine, lidocaine, etc.) are the most widely used drugs in PNBs. Single PNB only lasts several hours ([@B46]). Catheter-based PNBs using LA infusions can extend analgesia ([@B26]); however, their efficiency is limited by mechanical factors and can expose patients to large quantities of LA ([@B20]). Alternatively, several LA carriers with extended release have been developed, among which liposomes have been shown to be the least toxic ([@B42]). A multilamellar liposome formulation of bupivacaine (Exparel) has so far failed to show PNB of major nerves ([@B17]) and remains of limited indication ([@B41]). Further experimental work is required to understand the *in vivo* effects of liposomal LA nanomedicines.

*In vivo* rodent models for PNB have focused primarily on assessing behavioral responses following LA such as the motor performance on an inverted mesh ([@B25]), the tail flick latency to a thermal stimulus ([@B16]), or the vocalization responses to an electrical shock ([@B15]). Such sensory-motor behavioral techniques were successfully used to investigate the protracted effect of liposomal LA ([@B9]), and nevertheless, the effect on axonal function remains poorly investigated. In a previous study in human volunteers, we found that it is feasible to monitor the effect of LA *in vivo* by combining conventional conduction studies with nerve excitability studies by threshold-tracking ([@B33]). Here, we aimed to develop a translational mouse model where the same neurophysiological techniques can be used to investigate liposomal formulations of LA *in vivo*. To challenge the validity of the model, we tested an unilamellar liposomal formulation, consisting of a single lipid bilayer ([@B6]) which has faster release profile than multilamellar liposomes ([@B38]). The liposomes were filled with the LA lidocaine which has a shorter duration of action than bupivacaine in extended-release formulations ([@B19]).

Materials and Methods {#s1}
=====================

Mice, Anesthesia, and Experimental Design
-----------------------------------------

Investigations *in vivo* were carried out in adult mice of C57Bl background (3- to 5-month-old) with axonal expression of the Yellow Fluorescent Protein (YFP) reporter (homozygote Thy1-YFP) obtained from the Jackson Laboratory, Bar Harbor, ME, United States. General anesthesia was ensured using a 1:1 mixture of Hypnorm/Midazolam (5 mg/ml). A volume of 0.1 ml/10 g from the mixture was injected subcutaneously for induction, and then maintained with 50% hourly for up to 4 h as needed.

Using *in vivo* neurophysiological recordings (**Figure [1](#F1){ref-type="fig"}**), we monitored the recovery of tibial nerve conduction (**Figure [2](#F2){ref-type="fig"}**) and excitability (**Figure [3](#F3){ref-type="fig"}**) following injection of the LA. Slow subcutaneous injection of a volume of 50 μl at the ankle engulfed the tibial nerve in an easily observable bubble (**Figure [1C](#F1){ref-type="fig"}**) ensuring the reproducibility of the LA exposure without the need of ultrasound visualization as in the case of human PNBs ([@B33]). The monitoring of the systemic toxicity was carried out using a 1-channel electrocardiogram (ECG) recorded between the forepaws (**Figure [1B](#F1){ref-type="fig"}**) (MP150 with an ECG100C module, Biopac Systems Inc., United States).

![Experimental setup. **(A)** Tibial nerve fibers were stimulated proximally (PROX) at the sciatic notch of the ilium (sciatic nerve), to assess recovery of conduction across the local anesthesia (LA) site, and distally (DIST) to assess recovery of tibial nerve excitability. The evoked compound muscle action potential (CMAP) was recorded from the plantar muscles. **(B)** Needle electrode placement for PROX stimulation (S-, S+), CMAP recording (R1, R2), ground electrode (GND), and ECG recording (ECG-R, ECG-L); **(C)** Needle electrode placement for DIST stimulation (S-, S+), CMAP recording (R1, R2), and the ground electrode (GND). The detail at right depicts the local anesthetic injection needle (Inj) as well as the bubble engulfing the tibial nerve at the ankle. The baseline CMAP recordings evoked from the PROX and DIST stimulation sites were depicted in panels **(D,E)**, respectively. The amplitude CMAP was measured peak-to-peak (ppA) and the corresponding latency (Lat) to the first take-off from baseline. Note that from both stimulation sites, the evoked CMAP was followed by a secondary motor response (F-wave) resulting from antidromic activation of the motor neuron pool.](fnins-12-00494-g001){#F1}

![Development and recovery of conduction block after injection of the local anesthetic (LA). **(A)** The plantar compound muscle action potential CMAP evoked by proximal stimulation at the sciatic notch after non-encapsulated lidocaine (L. HCL) exposure. After 5 ms, the recordings are presented at 10-fold larger amplitude to facilitate the visualization of F-waves; **(B)** The relative changes in peak-to-peak CMAP amplitude (ppA) were shown for the non-encapsulated lidocaine (L. HCl) experiment in **(A)** versus 2 other different experiments using liposomal lidocaine (L. Lip); **(C)** ECG traces recorded during exposure to liposomal lidocaine. Note that the heart rate, measured from the QRS complexes, remained between 8 and 10 s (corresponding to 480--600 beats per minute). The ppA recovery corresponding to the ECG after 60 min is indicated in blue in panel **(B)**.](fnins-12-00494-g002){#F2}

![Recovery of excitability after injection of the local anesthetic (LA). Baseline measurements (PRE) are compared with measurements at 60 min after liposomal lidocaine (L. Lip) when there was a partial recovery of conduction. **(A)** Plantar compound motor action potentials (CMAPs) were evoked by distal stimulation of the tibial nerve at the ankle (within the nerve area exposed to LA). The thick traces indicate the CMAPs closest to 40% of the maximal CMAP amplitude, referred to as threshold CMAPs; **(B)** The stimulus-response measurements corresponding to the recordings in **(A)**. The stippled lines indicate the current required to evoke the threshold CMAP (symbols), referred to as threshold; **(C)** The multiple measures of nerve excitability by "threshold-tracking" protocol, measuring the changes in threshold when varying stimulus duration (charge--duration), when conditioning with 100 ms depolarizing (upward) and hyperpolarizing (downward) currents that were set to 40% of threshold (threshold electrotonus), when conditioning with a range of 200 ms polarizing currents that were stepped from +50% (top) to --100% (bottom) of threshold (current--threshold relationship) and following a supramaximal conditioning stimulus (recovery cycle). The arrows indicate that following liposomal lidocaine there was: an increase in the charge--duration slope, a reduction in threshold changes during both depolarizing and hyperpolarizing threshold electrotonus, a marked increase in the current--threshold slope on depolarization, and an increased in refractoriness of the recovery cycle.](fnins-12-00494-g003){#F3}

At the completion of the electrophysiological experiments, the lateral aspect of the leg was exposed for imaging studies (**Figure [4](#F4){ref-type="fig"}**) after which the mice were killed by cervical dislocation.

![Imaging of the tibial nerve block area by probe-based confocal laser endomicroscopy (pCLE) at 90 min after liposomal lidocaine (L. Lip). **(A)** The ankle was first dissected to allow access of the imaging probe (Probe) to the subcutaneous tissue. Note the scattering of the blue laser light used for imaging; **(B)** Superficial veins were fluorescent consistent with the absorption of the fluorescent liposomes. The arrow identifies the same venous confluence in light imaging and pCLE imaging; **(C)** With further advancement of the imaging probe between the muscle planes (blunt dissection), the tibial nerve could be readily identified due to its fluorescent fibers (axonal YFP reporter expression). Occasionally, liposomal accumulations (star) could be identified near the fibers.](fnins-12-00494-g004){#F4}

This study was carried out in accordance with the recommendations of directive 2010/63/EU of the European parliament and of the council on the protection of animals used for scientific purposes. The protocol was approved by the Danish Animal Experiments Inspectorate.

Electrophysiological Setup
--------------------------

For *in vivo* electrophysiological investigations, the mice were placed on a temperature-controlled pad (HB 101/2, LSI Letica) set to 37°C ([@B32]). Stimulation and recording were carried out using percutaneously inserted Pt-needle electrodes ([@B32]; [@B44]). Electrical stimuli generated from a constant current stimulator (DS4, Digitimer Ltd.) were delivered proximally to the sciatic nerve at the sciatic notch (**Figures [1A,B](#F1){ref-type="fig"}**) and distal to the tibial nerve at the ankle (**Figures [1A,C](#F1){ref-type="fig"}**). The evoked compound muscle potential (CMAP) of the plantar muscle was recorded using a bandpass filter of 10 Hz--6 kHz (Neurolog NL820A amplifier with NL844 Pre-Amplifier, Digitimer Ltd., United Kingdom). A ground reference electrode was inserted into the back of the mouse. The near-nerve electrode placement was carried out ensuring the lowest threshold current.

The signals were digitized into a PC-based recording and control system via a multifunction I/O device (PCI-6221, National Instruments Corporation Ltd., United Kingdom). The amplitudes of the CMAP evoked from the proximal (**Figure [1D](#F1){ref-type="fig"}**) and distal (**Figure [1E](#F1){ref-type="fig"}**) stimulation sites were measured peak-to-peak (ppA). The CMAP latency was measured to the first take-off from baseline (**Figures [1D,E](#F1){ref-type="fig"}**).

It should be noted that both from the proximal stimulation site (**Figure [1D](#F1){ref-type="fig"}**) as well as from the distal stimulation site (**Figure [1E](#F1){ref-type="fig"}**), the evoked CMAP was followed by a secondary motor response ("F-wave") resulting from motoneuronal backfiring following antidromic activation in humans ([@B27]) and rodents ([@B28]; [@B35]; [@B31]). Although the F-waves were not specifically quantified, their presence was considered as an indication of the functional integrity of the motoneurons and the alpha-motor fibers.

Nerve Conduction Studies
------------------------

We have previously established a setup for the continuous monitoring of tibial nerve conduction by stimulation at the ankle for up to 4 h ([@B32]; [@B2]). Nevertheless, overcoming the partial voltage-gate Na+ channel block by lidocaine could require very large stimulation currents ([@B33]). Given the small distances, using such high currents at ankle could cause the stimulus to" jump" and activate the unanesthetized part of the tibial nerve. To avoid this potential confounder, we tested the changes in conduction across the PNB by proximal stimulation (**Figures [1A,B](#F1){ref-type="fig"}**). Stimulation and recording were controlled using a custom control software developed in MATLAB (version R2013b, MathWorks Inc., United States). We delivered negative rectangular stimuli of 1 ms duration at 0.5 Hz and recorded 1000 ms sweeps with a sampling frequency of 125 KHz allowing synchronized CMAP (**Figure [2A](#F2){ref-type="fig"}**) and ECG recordings (**Figure [2C](#F2){ref-type="fig"}**). The amplitudes at proximal stimulation were averaged over 1-min and expressed, relative to the 1st minute prior to injection (**Figure [2B](#F2){ref-type="fig"}**). The stimulation current was set at twofold the current required to obtain a 100% ppA CMAP and kept constant for the entire duration of the recording.

Nerve Excitability Studies
--------------------------

The changes in nerve excitability at the PNB site were monitored by distal stimulation at the ankle (**Figures [1A,C](#F1){ref-type="fig"}**) and tracking changes in the threshold current (**Figures [3A,B](#F3){ref-type="fig"}**) required to evoke a 40% ppA CMAP ([@B7]) using QtracS stimulation software for recording and QtracP software for analysis (©Institute of Neurology, London, United Kingdom). We used that same TRONDH multiple excitability protocol ([@B22]), that we used in our human PNB study ([@B33]). We have previously adapted this protocol for use in mice ([@B29], [@B30]). This allowed recording of the full sequence of excitability measures (**Figure [3C](#F3){ref-type="fig"}**) as previously described in detail ([@B7]; [@B22]; [@B23]): charge--duration relationship to measure the rheobase, threshold electrotonus to measure accommodation to shifts in depolarizing and hyperpolarizing membrane potential, current--threshold relationship to measure input conductance, and the recovery of excitability following conduction of the action potential to measure refractoriness.

Imaging Studies
---------------

*In vivo* imaging (**Figure [4](#F4){ref-type="fig"}**) was carried out using probe-based confocal laser endomicroscopy (pCLE) using a Cellvizio single band, with a 488-nm laser (Mauna Kea Technologies, France) which allows for the investigation of fluorescent blood vessels and axons ([@B45]). The frames were captured via a S-1500 objective (**Figure [4A](#F4){ref-type="fig"}**) and exported using the Cellvizio Viewer for PC (version 1.4.2).

Liposomal LA Formulation
------------------------

We formed an unilamelar liposomes using the established methods of freeze drying lipids followed by rehydration in lidocaine hydrochloride (2% w/v) phosphate-buffered saline. This solution was pressure extruded through 100 nm filters to reduce liposome size polydispersity. The liposomes comprised of hydrogenated soy L-α-phosphatidylcholine (HSPC), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 (DSPE-PEG2000), and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine (TopFluor PC) in a mol% ratio of 54.8:40:5:0.2, respectively. Polyethylene glycol conjugated lipid was introduced into the liposome bilayer to increase residency time ([@B10]) and TopFluor PC was incorporated to allow visualization. The resulting liposome size (Mean ± SD) was found to be 196.2 ± 2.2 nm with a size polydispersity index of 0.134 ± 0.03 as determined by dynamic light scattering. A non-encapsulated lidocaine salt solution (20 mg/ml, L. HCl) served as control. All chemical components were purchased from commercial suppliers (Sigma--Aldrich, DE, and Avanti Polar Lipids, United States).

Results
=======

Recovery of Conduction
----------------------

Proximal stimulation of the tibial nerve fibers at the sciatic notch (**Figures [1A,B](#F1){ref-type="fig"}**) evoked a CMAP (Mean ± SD) with a ppA of 12 ± 4 mV and a latency of 1.7 ± 0.1 ms (**Figure [1D](#F1){ref-type="fig"}**). Distal stimulation of the tibial nerve fibers at the ankle (**Figures [1A,C](#F1){ref-type="fig"}**) evoked a CMAP (Mean ± SD) with a ppA of 16 ± 5 mV and a latency of 1.2 ± 0.1 ms (**Figure [1E](#F1){ref-type="fig"}**). Note that the proximal CMAP was not larger than the distal CMAP indicating that the CMAP measurements reflected tibial-nerve innervated muscles with minimal contamination (if any) from common peroneal nerve innervated muscles.

An experiment with non-encapsulated lidocaine is detailed in **Figure [2A](#F2){ref-type="fig"}**. Within 3 min following LA injection, there was a complete abolishment of the CMAP and F-waves evoked by proximal stimulation which recovered completely within 30 min (**Figure [2A](#F2){ref-type="fig"}**). The time-course of CMAP amplitude (ppA) recovery, measured relative to the minute before LA, is presented in **Figure [2B](#F2){ref-type="fig"}**. In contrast, our model was able to detect a slower recovery after liposomal lidocaine, with good reproducibility in repeated studies (**Figure [2B](#F2){ref-type="fig"}**). Furthermore, we found that following liposomal lidocaine the CMAP evoked by proximal stimulation recovered rapidly to above 50% ppA whereas the subsequent recovery was much slower (**Figure [1B](#F1){ref-type="fig"}**), so that a full recovery was observed after 3 h (data not shown). Although liposomes were absorbed, as indicated by pCLE (**Figure [4B](#F4){ref-type="fig"}**), there were no ECG signs of bradycardia (**Figure [2C](#F2){ref-type="fig"}**) that could indicate a confounding effect of lidocaine toxicity.

Recovery of Excitability
------------------------

During the slow CMAP recovery phase after liposomal lidocaine (**Figure [2B](#F2){ref-type="fig"}**), liposomal accumulations could be demonstrated by pCLE near the tibial nerve fibers at the ankle (**Figure [4C](#F4){ref-type="fig"}**) suggesting a persistent release. An experiment with uncaged lidocaine is detailed in **Figure [3](#F3){ref-type="fig"}**. We found that during the partial CMAP recovery phase, evoking the maximal CMAP by distal stimulation in the nerve exposed to LA required a much larger stimulation current (**Figure [3A](#F3){ref-type="fig"}**) with a right-shift in the stimulus-response curve (**Figure [3B](#F3){ref-type="fig"}**). At that time, the current required to evoke the CMAP from the proximal stimulation site remained undistinguishable from the value prior to LA (data not shown).

To explore the mechanisms of impaired tibial nerve excitability at the LA site, we carried out multiple measures of excitability by "threshold-tracking" (**Figure [3C](#F3){ref-type="fig"}**). Consistent with the larger stimulus current required to evoke the CMAP, we found a marked increase in rheobase ([@B43]; [@B7]), as indicated by the slope of the charge--duration relationship (**Figure [3C](#F3){ref-type="fig"}**). Moreover, the observed increase in rheobase after liposomal lidocaine was associated with (**Figure [3C](#F3){ref-type="fig"}**) a reduction in threshold changes during both depolarizing and hyperpolarizing threshold electrotonus, a marked increase in the current--threshold slope (input conductance) on depolarization, and an increased in refractoriness of the recovery cycle (**Figure [3C](#F3){ref-type="fig"}**).

Discussion
==========

We developed an *in vivo* mouse model to explore the effect of local anesthetic nanomedicines on axonal function. We tested the validity of the model by comparing the effect of a liposomal lidocaine formulation versus non-encapsulated lidocaine on motor axon function and found a slower conduction recovery along the tibial nerve following liposomal lidocaine. Furthermore, the model opened the possibility to combine conduction studies with "threshold-tracking" excitability studies in the same nerve, allowing a translational mechanistic insight into the liposomal release *in vivo*.

Assessment of the effect of liposomal local anesthetic nanomedicines on sciatic nerve block in rodents has been previously carried out previously by behavioral measures ([@B47]). The sensory (thermal nociceptive) blockade was investigated in a hot-plate paradigm ([@B36]), whereas the motor blockade was investigated using the extensor postural thrust test ([@B36]). Although these behavioral studies provided a valuable approach to distinguish the motor versus sensory selectivity of the nanomedicine, they offered little information on the actual changes in axonal function. It is long known that the susceptibility to an anesthetic conduction block is different between neuronal populations, reflecting differences in membrane properties ([@B12]). Nevertheless, motor population was found to be at least as good an indicator for changes in the duration of PNB as the sensory fibers ([@B8]; [@B14]) even though they do not directly reflect the analgesic effect. Furthermore, the fact that the myelinated motor axon population is more functionally homogenous than the sensory fiber population ([@B12]), can convey an advantage in characterizing the excitability changes.

The LA lidocaine is thought to impair the function of the voltage-gated Na^+^ channels (VGSC) ([@B37]) reducing the axonal "safety factor" for conduction ([@B40]) in excitable tissues of the nervous system ([@B34]) as well as the heart ([@B4]). Although the pCLE imaging studies indicated liposome accumulation within veins (**Figure [4B](#F4){ref-type="fig"}**), the ECG (**Figure [2C](#F2){ref-type="fig"}**) remained within normal limits ([@B18]). We therefore do not think that the observed effects on axonal function were confounded by lidocaine cardiotoxicity ([@B11]).

We confirmed an increase in the threshold for electrical stimulation during recovery after LA ([@B12]; [@B13]). Similar deviations in excitability were found in our previous study using non-encapsulated lidocaine in humans ([@B33]) which supports the translational value of our mouse model. These excitability deviations differed from those observed with selective VGSC block following accidental tetrodotoxin poisoning in humans ([@B24]). It is likely that the protracted period of reduced nerve excitability following LA ([@B39]) reflected both the partial recovery of the Na^+^ conductance as well as an impairment of the axolemmal passive electrical properties ([@B33]) due to a physical effect of lidocaine dissolved within the membrane ([@B21]). In further studies, excitability changes could allow the detection of early signs of toxicity ([@B29]) in combination with imaging of morphological changes ([@B5]; [@B2], [@B3]).

Author Contributions
====================

CK and KL initiated the study. MM made the neurophysiological control software. AU made the liposomal lidocaine formulation. All the authors contributed to the experiments, analyzed the data, discussed the results, and wrote the manuscript.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** The project was supported by the Danish Medical Research Council grants (4092-00100/2014 and 7016-00165/2017), the Lundbeck Foundation, and the Foundation for Research in Neurology and Jytte and Kaj Dahlboms Foundation.

[^1]: Edited by: Ioan Opris, University of Miami, United States

[^2]: Reviewed by: Angelika Lampert, Uniklinik RWTH Aachen, Germany; Mario Valentino, University of Malta, Malta

[^3]: This article was submitted to Neural Technology, a section of the journal Frontiers in Neuroscience
