j vaa 2020 07 036

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Evaluation of a regional nerve block with an experimental formulation of encapsulated
lidocaine in sheep
Erika A. Militana, Luis Campoy, Manuel Martin-Flores, Robin D. Gleed
PII: S1467-2987(20)30177-X
DOI: https://doi.org/10.1016/j.vaa.2020.07.036
Reference: VAA 545
To appear in: Veterinary Anaesthesia and Analgesia
Received Date: 22 April 2020
Revised Date: 9 July 2020
Accepted Date: 28 July 2020
Please cite this article as: Militana EA, Campoy L, Martin-Flores M, Gleed RD, Evaluation of a
regional nerve block with an experimental formulation of encapsulated lidocaine in sheep, Veterinary
Anaesthesia and Analgesia, https://doi.org/10.1016/j.vaa.2020.07.036.
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© 2020 Published by Elsevier Ltd on behalf of Association of Veterinary Anaesthetists and American
College of Veterinary Anesthesia and Analgesia.
https://doi.org/10.1016/j.vaa.2020.07.036
https://doi.org/10.1016/j.vaa.2020.07.036
RESEARCH PAPER 
Running head (Authors) EA Militana et al. 
Running head (short title) Encapsulated lidocaine 
Evaluation of a regional nerve block with an experimental formulation of encapsulated 
lidocaine in sheep 
Erika A Militana, Luis Campoy, Manuel Martin-Flores & Robin D Gleed 
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, 
NY, USA 
 
Correspondence: Luis Campoy, Department of Clinical Sciences, College of Veterinary 
Medicine, Cornell University, 602 Tower Rd, Ithaca, NY 14853, USA. E-mail: 
luis.campoy@cornell.edu 
 
 
Abstract 
Objective To compare the duration of nociceptive and proprioceptive blockade from an 
experimental encapsulated lidocaine preparation with that of conventional lidocaine. 
Study design Prospective, blinded, randomly assigned, crossover study. 
Animals A total of six adult ewes, American Society of Anesthesiologists physical status I or II, 
weighing 60.4 ± 18.0 kg (mean ± standard deviation). 
Methods Under general anesthesia and guided by electrolocation, the common peroneal nerve 
was blocked unilaterally with encapsulated lidocaine (0.1 mL kg−1, 200 mg mL−1) or 
conventional lidocaine hydrochloride (0.1 mL kg−1, 20 mg mL−1). Each sheep was administered 
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both treatments with an interval between treatments of 2 weeks. Nociception and proprioception 
were scored (scales of 0–3) before anesthesia, at 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours after 
completion of local anesthetic injection, and every 12 hours thereafter, for 9 days. Nociceptive 
and proprioceptive blockade ended the first time each score reached ‘0’; maximum blockade 
duration was considered and recorded to be the time point immediately prior to this end point. 
Significance of differences between treatments for duration of blockade was tested with the 
Wilcoxon rank-sum test. Effects of time and treatment on nociceptive and proprioceptive 
blockade were evaluated with mixed-effect models. Significance was set at padjusted (within the range of 50–100 µm) so 
that they released continuously for 7 days. At the time of injection, the suspension contained 200 
mg mL−1 of lidocaine, 94% of which was contained within the microcapsules while the balance 
was free in the vehicle. 
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Under the Cornell University Institutional Animal Care and Use Committee approval 
(2006-0148), six adult Dorset ewes American Society of Anesthesiologists physical status I or II 
and weighing 60.4 ± 18.0 kg [mean ± standard deviation (SD)] were used. Each sheep was 
anesthetized twice with 2 weeks between anesthetic episodes. The encapsulated lidocaine 
preparation (Encap, 200 mg mL−1; treatment Encap) was tested in one episode and 2% lidocaine 
hydrochloride (Lidocaine hydrochloride USP, 20 mg mL−1; Hospira Inc., IL, USA; treatment 
LidoHCl) was tested in the other. For each sheep, the order of the two treatments was assigned 
randomly by withdrawing ballots from a closed envelope; the envelope contained three ballots 
indicating that treatment Encap should be first, and another three indicating that treatment 
LidoHCl should be first. During the first anesthetic episode, one peroneal nerve was blocked; 
during the second episode, the contralateral peroneal nerve was blocked. 
The animal was sedated with midazolam (0.2 mg kg−1; Midazolam hydrochloride; 
Hospira Inc.) intravenously (IV) and approximately 10 minutes later, anesthesia was induced 
with IV propofol (PropoFlo; Abbott Laboratories, IL, USA) until consciousness was lost, 
swallowing reflex was absent and laryngeal abduction was satisfactory to achieve orotracheal 
intubation. Isoflurane (Isoflurane USP; Phoenix Pharmaceutical Inc., MO, USA) was 
administered in oxygen via a circle partial rebreathing system connected to the endotracheal 
tube. The lungs were mechanically ventilated to achieve an end-tidal partial pressure of carbon 
dioxide of 35–40 mmHg (4.5–5.5 kPa). The isoflurane concentration was adjusted to ensure 
unconsciousness and immobility. 
Peroneal nerve blocks were performed using a standard electrolocation-assisted 
technique, similar to a previous description in dogs (Campoy et al. 2012). The peroneal nerve 
block was located with the animal in lateral recumbency, with the limb to be blocked uppermost 
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and extended in a natural position. The puncture site was identified in the groove formed by the 
peroneus longus and lateral digital extensor muscles distal to the stifle joint. 
The technique was performed using a stimulating needle (18 gauge, 5 cm Contiplex 
Tuohy; B Braun Medical Inc., PA, USA) connected to a peripheral nerve locator (Innervator 
232; Fisher & Paykel Healthcare Ltd., Auckland, NZ) at an initial output of 1 mA, frequency of 
1 Hz and duration of 0.1 ms for both treatments. When the tip of the needle was located in close 
proximity to the peroneal nerve, dorsiflexion of the foot was elicited. Injection was performed 
around the nerve when muscular contractions were elicited at 0.4 mA but not at 0.2 mA, and 
following negative blood aspiration and absence of resistance to injection. The same individual 
(LC), unaware of treatment allocation, performed all injections. 
Each sheep was recovered from anesthesia immediately after completion of each 
injection and then returned to the pen where it had been previously housed. Nociceptive and 
proprioceptive function were scored before anesthesia, at 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours 
after recovery from general anesthesia, and every 12 hours thereafter for 9 days by an 
investigator who was unaware of treatment allocation (RDG) (Appendices A & B). Nociceptive 
function was scored based on responses to application of mosquito forceps to the skin just 
proximal to the craniolateral aspect of the coronary band. These responses included conscious 
limb retraction accompanied by head turning or postural change. The concomitant response of a 
head turn or posture change helped distinguish a conscious limb retraction from a withdrawal 
reflex. 
An over-the-needle catheter (14 gauge, 13 cm Teflon catheter; Mila International Inc., 
KY, USA) was placed in the external jugular vein of one sheep undergoing each treatment, such 
that blood samples were collected from the same sheep on two occasions. A three-syringe 
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technique was used for blood collection: the first syringe was attached to the catheter port and 
used to aspirate a volume of fluid (blood and heparinized saline) equal to 10 times the volume of 
the catheter, the second syringe was used to withdraw the blood sample (2 mL) and the contents 
of the first syringe were injected back into the catheter, and a third syringe containing 
heparinized saline (2 units mL−1; Heparin Sodium; B Braun Medical Inc., CA, USA) was used to 
flush the catheter. Blood samples were collected before treatment injection, at 1, 4 and 24 hours 
after completion of local anesthetic injection, and every 24 hours thereafter for 7 days. 
Immediately after collection, the sample was placed in a glass blood collection tube containing 
5.4 mg of K2-ethylenediamine tetra-acetic acid (EDTA BD Vacutainer; NJ, USA) and rotated 
gently by hand 10 times to prevent clotting. The sample was then centrifuged at room 
temperature for 10 minutes (674 g; Heraeus Labofuge 200; Thermo Fischer Scientific Inc., MA, 
USA). The resultant plasma was removed using a pipette and added to a cryovial stored at –28 
oC until delivered for analysis (NMS Labs; PA, USA). Lidocaine concentrations were measured 
by gas chromatography with a lower reporting limit of 0.50 µg mL−1, similarly to a previously 
reported technique (Watanabe et al. 1998). 
The sheep were euthanized by IV barbiturate overdose (100 mg kg−1; Fatal Plus; Vortech 
Pharmaceuticals Ltd, MI, USA) at the completion of the study, 10 days after the second 
injection. Immediately after euthanasia, the peroneal nerve and surrounding tissues were 
dissected bilaterally and examined for gross evidence of pathology at the injection site. The 
peroneal nerve at the site of injection and the regional lymph nodes were harvested bilaterally 
and processed for histological examination by a board-certified pathologist who was unaware of 
which treatment was associated with a sample. 
 
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Statistical analysis 
A sample size of three animals was calculated (G*Power 3.1.5; Universität Kiel, Germany) with 
α set at 0.05, β set at 0.95, and assuming normal distribution of data. Nociception duration was 
assumed to be 72 ± 12 hours (mean ± SD) in the Encap treatment and 5 ± 2 hours (mean ± SD) 
in the LidoHCl treatment (based on unpublished pilot data). The sample size software was set to 
run a paired-sample, one-tailed test. The sample size was doubled to allow for possible loss of 
data or uncooperative animals. 
After treatment, nociceptive and proprioceptive blockade were recorded to have ended 
the first time each score reached ‘0’; however, the maximum duration of blockade was 
considered to be the time point immediately prior to the time point when scores first reached 0. 
The significance (pLidoHCl (p = 0.008; Table 1). Median duration of proprioceptive blockade 
was 6 hours longer in Encap than LidoHCl (p = 0.03; Table 1). Although the median Encap 
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nociceptive blockade was 90 hours, one Encap-treated animal was an outlier with a nociceptive 
blockade that lasted only 4 hours (Table 1); proprioceptive block lasted 4 hours in the same 
animal. 
Significant effects of time (p 11.7 
± 2 µg mL−1 produce adverse effects in sheep (Morishima et al. 1990), it is reassuring that the 
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lidocaine concentration in plasma after Encap (and LidoHCl) never exceeded 0.50 µg mL−1 in 
the one sheep in which systematic measurements were made. 
A major limitation of this study is the sample size, even though significant differences 
were detected. Despite a power analysis suggesting that the sample size was adequate to detect 
differences in median blockade duration, it was not designed to identify the frequency of certain 
findings such as occurrence of prolonged proprioceptive deficits. These observations should 
encourage the use of measures to detect and prevent complications associated with long-term 
proprioceptive deficits, such as abrasions, especially when encapsulated preparations are 
selected. In one of the animals in Encap, apparent conduction blockade was short for both 
nociception and proprioception. The possibility that the test suspension was deposited farther 
from the nerve than is optimal in this sheep cannot be excluded. Accurate perineural blockade is 
technically difficult and subject to occasional failure, although other techniques, such as 
ultrasound guidance, might reduce the failure rate. In addition, the experimental preparation was 
viscous, prone to aggregation, and therefore difficult to inject through needles with an internal 
diameter smaller than that of an 18 gauge needle. Ongoing assessment to confirm that 
nociception blockade is effective in clinical patients would be justified, especially when direct 
visualization of the target nerve and subsequent deposition of local anesthetic is precluded. 
The sheep nociception model is difficult due to the stoic nature of the animal, causing 
some degree of uncertainty in correctly identifyingpain. To address this limitation, and in an 
attempt to add objectivity, a nociceptive withdrawal score was implemented, which was adapted 
from a sheep model based on the use of a short-acting electrical stimulus to evoke a withdrawal 
reaction accompanied by a nocifensive behavioral reaction (Rohrbach et al. 2014). Although the 
current descriptors for nociceptive blockade were not used or described previously in response to 
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a mechanically noxious stimulus such as mosquito forceps, the grading system is similar to the 
behavioral reaction described for electrical stimulation. Furthermore, variability in the results 
was in part minimized by blinding, randomization and the crossover design that allowed each 
sheep to serve as its own control. 
 
Conclusions 
These findings suggest a clinical use for blocking peripheral nerves with PLGA-encapsulated 
sustained-release lidocaine. Regional anesthesia as an adjunct to general anesthesia may 
markedly improve postoperative pain control and reduce the reliance on systemic analgesics. 
 
 
Acknowledgements 
The authors thank Capsulated Systems Inc. for donating the study drug. 
 
Author’s contributions 
EAM: database search, data interpretation, preparation of manuscript, manuscript review. LC: 
study design, data collection, data interpretation, manuscript review. MMF: study design, 
statistical analysis, data interpretation, manuscript review. RDG: study design, manuscript 
review. All authors read and approved the final version of the manuscript. 
 
Conflict of interest statement 
The authors declare no conflict of interest. 
 
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Figure 1 (a) Nociceptive and (b) proprioceptive scores in six sheep administered 
perineural injection of the peroneal nerve with encapsulated lidocaine (treatment Encap) or 
conventional 2% lidocaine (treatment LidoHCl) before (time 0) and after recovery from general 
anesthesia. Data are mean ± standard error of the mean. Data at 0 and 0.5 hours are not shown 
for either treatment for clarity. 
 
 
Figure 2 Dissection of the lateral aspect of a pelvic limb in a sheep 10 days after injection 
around the peroneal nerve with encapsulated lidocaine. Injection site is identified by the 
arrowhead. No gross abnormalities of the nerve or surrounding tissue were detected. 
 
 
Figure 3 Histological preparation of peroneal nerve perineural tissue obtained from a sheep 10 
days after injection of encapsulated lidocaine and stained with hematoxylin and eosin. 
Phagocytosed microcapsules (arrowhead) can be seen within the clear cytoplasmic vacuoles 
(magnification ×100). 
 
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Appendix A Nociceptive blockade scoring system. 
Score 
0 
Description 
No block – apparently instant response to applying mosquito forceps 
1 Delayed response to applying mosquito forceps 
2 Almost able to clamp mosquito forceps to first ratchet 
3 No response even after first ratchet of the mosquito forceps 
 
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Appendix B Proprioceptive blockade scoring system 
Score 
0 
Description 
No block – no fetlock joint knuckling observed even when chased 
1 Some impairment, occasional fetlock joint knuckling when chased 
2 Fetlock joint knuckling when standing, but able to achieve normal position 
3 Persistent fetlock joint knuckling while standing 
 
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Table 1 Duration (hours) of nociceptive and proprioceptive blockade [median (range)] in 
six sheep after perineural injection of encapsulated lidocaine (treatment Encap) or 
conventional lidocaine (treatment LidoHCl) for peroneal nerve block. Nociceptiveand 
proprioceptive blockades ended for each sheep the first time the scores were ‘0’, with 
maximum duration blockade considered to be the time point immediately prior to this 
observation period. 
Parameter 
Treatment 
p 
Encap LidoHCl 
Nociception 
(hours) 90 (4–180) 2 (1–8) 0.008 
Proprioception 
(hours) 8 (1–72) 2 (1–4) 0.03 
 
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0
1
2
3
36 60 84 108 132 156 180 204
Time (hours)
P
ro
p
io
ce
p
ti
v
e
sc
o
re
(0
-3
)
Encap
LidoHCl
1 2 4 8 1216 20 24
(b)
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0
1
2
3
36 60 84 108 132 156 180 204
Time (hours)
N
o
ci
ce
p
ti
v
e
sc
o
re
(0
-3
)
Encap
LidoHCl
1 2 4 8 1216 20 24
(a)
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