Micr 30482

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Received: 5 March 2019 Revised: 20 May 2019 Accepted: 24 May 2019

DOI: 10.1002/micr.30482

RESEARCH ARTICLE

The rabbit brachial plexus as a model for nerve injury and


repair Part 1: Anatomic study of the biceps and triceps
innervation

Kathleen M. Kollitz MD | Guilherme Giusti MD | Patricia F. Friedrich AAS |


Allen T. Bishop MD | Alexander Y. Shin MD

Department of Orthopedic Surgery, Division


of Hand and Microvascular Surgery, Abstract
Mayo Clinic, Rochester, Minnesota Purpose: Animal models can be helpful in evaluating new surgical strategies for
Correspondence brachial plexus reconstruction. While several groups have already used the rabbit
Alexander Y. Shin, MD, Department of brachial plexus to model injury, reports conflict in anatomic detail and do not identify
Orthopaedic Surgery, Mayo Clinic,
200 1 St SW, Rochester, MN 55905. a nerve-muscle pair to measure motor function recovery after reconstruction. The
Email: shin.alexander@mayo.edu purpose of the current study is to describe the innervations of the biceps and triceps
Funding information muscles in rabbits, which are both amenable to study in brachial plexus injury models.
American Foundation for Surgery of the Hand Materials and Methods: Thirteen rabbits weighing 2–2.5 kg were anesthetized. Six
rabbits were sacrificed and dissected using loupe and microscope magnification to
understand the overall morphology of the brachial plexus. Seven rabbits underwent
electrophysiologic investigation. A bipolar nerve stimulator was used to systemati-
cally stimulate the roots, trunks and divisions, and nerve branches of the rabbit bra-
chial plexus and compound muscle action potential was used to record muscle
response. Nerve length and width measurements were not recorded.
Results: Roots contributing to the brachial plexus were C5, C6, C7, C8, and T1. In
contrast to other anatomical studies, T2 did not contribute to the brachial plexus.
The triceps was innervated by the radial nerve, which received contributions from C6
(1.6 mA), C7 (1.9 mA), C8, and T1 (12.2 mA).The biceps had dual innervation (proxi-
mally and distally). The proximal branch received contributions from C6 (3.5 mA) and
C7 (5mA). The distal portion was innervated by a branch from the median nerve,
which received innervation from C6, C7, C8, and T1.
Conclusions: The overall structure of rabbit brachial plexus is described and innerva-
tion of the biceps and triceps is described in detail. This anatomic investigation will
form the basis of a future brachial plexus model of injury and repair.

1 | I N T RO D UC T I O N outcomes remain variable, and alternatives to nerve autograft and


transfer would address these clinical challenges.
Surgical treatment of brachial plexus injuries can offer functional Given the complexity of options available for reconstruction and
recovery, though the options are many and the optimal strategy the variability seen in outcomes, there is a need for animal models of
remains controversial. Complex injuries to the brachial plexus are brachial plexus injury to help develop and evaluate new surgical treat-
often reconstructed through a combination of nerve graft, nerve ments. Experimental models of brachial plexus injury have been
transfer, free functioning muscle transfer, and tendon transfer. Clinical largely limited to the rat and mouse (Ibrahim, Raisman, & Li, 2009;

Microsurgery. 2019;1–6. wileyonlinelibrary.com/journal/micr © 2019 Wiley Periodicals, Inc. 1


2 KOLLITZ ET AL.

Kim, Galatz, Patel, Das, & Thomopoulos, 2009; Kostopoulos, 2.2 | Electrophysiology
Konofaos, Frazer, & Terzis, 2010; Liu et al., 2015; Riva et al., 2012;
Seven rabbits underwent a nonsurvival surgical procedure to map out
Wang, Spinner, & Windebank, 2009; Yang et al., 2015; Yang, Yang,
electrophysiology of the biceps and triceps muscles. Anesthesia was
Yu, & Gu, 2014). The outcomes of reconstruction in rodents are uni-
induced with an intramuscular injection of Ketamine 35 mg/kg,
versally good secondary to the exceptional regenerative capacity of
Xylazine 5 mg/kg, and Buprenorphine 0.03/kg. The neck, chest, and
rodents, and relatively short gap that can be created (Wang et al.,
upper extremity were clipped of hair. Rabbits were intubated and
2009). The rabbit has also been proposed as an experimental model anesthesia was maintained with intravenous propofol. Lactated
for brachial plexus injury (Cao, Li, Cao, & Cai, 2003; Kawai et al., Ringers solution was infused at 5 cc/hr to maintain hydration. Tem-
1989; Mohiuddin, Rahman, Alim, Kabir, & Kashem, 2011; Reichert perature was checked by a rectal thermometer and maintained with a
et al., 2014, 2015; Stamatoukou et al., 2006; Zhang et al., 2006). Rab- heating pad. At the end of the procedure, animals were sacrificed with
bits do not appear to have supranormal healing potential, and offer an overdose of Phenobarbital IV while they were still anesthetized.
cost advantage as compared to larger animals such as nonhuman pri- Rabbits were positioned supine. The right and left sides were
mates, pigs, sheep, or dogs, for example. Existing studies of the rabbit alternately used for the initial stimulation study. A 5 cm curvilinear
brachial plexus have described the structure of the brachial plexus shaped incision was made from the neck passing lateral to the sterno-
and nerve measurements and histomorphometry. For example, clavicular joint, along the clavicle and to the deltopectoral interval.
Reichert et al. (2014) have previously reported the average diameter Cranially, the sternocephalus and cleidocephalus muscles were

of the nerve roots forming the brachial plexus as follows: divided to expose the jugular vein, C5 and C6 nerve root. The clavicle

C5—0.6 mm; C6—0.8 mm, C7—1.1 mm, C8—1.0 mm, T1—0.6 mm. At was divided at its ligamentous attachment from the sternum and the
pectoralis muscle was divided from its origin on the sternum; these
the trunk level, they reported the cranial truck as 1.2 mm, the medial
were retracted caudally to expose the C7 nerve root and lower trunk.
portion of the caudal trunk (or “middle trunk”) as 1.3 mm, and the lat-
Using bipolar needle stimulation, the roots were systematically iden-
eral portion of the caudal trunk (“lower trunk”) as 1.6 mm (Reichert
tified and stimulated with 0.7 mV for 0.02 ms where they exited the
et al., 2014). They identified the brachial plexus as appropriate for
neck musculature. C8/T1 united prior to exiting the neck musculature,
microsurgical intervention through this work. The next step in devel-
and were therefore tested as a group. The lowest voltage and shortest
oping a rabbit model of motor functional recovery would be to iden-
stimulation time were used to minimize signal spread throughout the
tify a nerve-muscle pair which could be used in hypothesis-driven
plexus. Compound muscle action potentials (CMAP) were recorded in
research of brachial plexus reconstructive options. The specific course
the biceps and triceps muscles. Contributions to the innervation were
of the terminal nerve branches for the biceps and triceps have not
then severed and stimulation was again performed. The contralateral
previously been described. Similarly, electrophysiology has not previ- side was then examined in the same way. After sacrifice, the plexus was
ously been used to verify innervation patterns. examined for similarity in structure to that of the Dutch Belted breed.
The goal of the current study is to build on previous work and
describe the anatomy and innervation of the rabbit biceps and triceps
using both anatomical observation and electrophysiology with the
2.3 | Statistical analysis
goal of establishing a nerve-muscle pair in which to study motor CMAP averages for biceps and triceps along with 95% confidence
recovery in an intraplexal injury model. intervals were calculated.

2 | MATERIALS AND METHODS 3 | RESULTS

Thirteen rabbits were used in this nonsurvival experiment which was No significant anatomical differences between Dutch Belted and
approved by our Institutional Animal Care and Use Committee. New Zealand White rabbits were identified.

2.1 | Anatomic dissection 3.1 | Surgical landmarks


Six male Dutch Belted rabbits weighing 2–2.5 kg were sacrificed with As a result of extensive dissection and observation, the following sur-
an overdose of phenobarbital (FatalPlus, Vortech Pharmaceuticals, gical landmarks were used to identify nerve roots through a more min-
Dearborn, MI) and dissected using loupe and operating microscope imal approach. The C6 nerve root was identified first during a
magnification to obtain an overall understanding of the structure of supraclavicular exposure, located just cranial to the clavicle. It was
the brachial plexus. Photographs were taken, and drawings were pro- easily recognized by its early branching contributions to C7. C7 was
duced. Wires were inserted along the nerve root into the neural reliably found just deep to the clavicle, cranial to the subclavian artery.
foramina and radiographs were obtained to accurately identify the A vein was variably found draped over this nerve root, and was dis-
nerve roots contributing to the rabbit brachial plexus. sected free and ligated when present. The lower trunk, consisting of
KOLLITZ ET AL. 3

the C8 and T1 nerve roots was identified between the subclavian vein contribute to the brachial plexus in any animal. The rabbit brachial
and artery, and was carefully dissected free from these vascular struc- plexus exhibited a common gross morphology (Figure 3). Visual
tures. C8 and T1 joined to form the lower trunk immediately after inspection revealed that C6 contributes to C7, which forms a robust
exiting from the musculature of the neck, near the junction of the sub- trunk with many branches (Figure 4).
clavian vein, and the jugular vein. These landmarks are illustrated in In the rabbit brachial plexus, C5 contributed a branch to C6 but
Figure 1. otherwise did not contribute to any muscles in the upper extremity.
Nerve roots contributing to the brachial plexus included C5, C6, C6 continued on to form the upper trunk which innervated the mus-
C7, C8, and T1 (Figure 2). The T2 nerve root was not observed to cles of the rotator cuff. We observed C6 joining C7 which again
branched, giving off a nerve to biceps which entered the muscle proxi-
mally. The middle trunk also contributed to the median nerve. The
median nerve in turn gave off a branch to the biceps more distally
(Figure 5).
The lower trunk (C8, T1) joined with a branch from the middle
trunk (C6, C7) to form the radial nerve, which wrapped around the
humerus posteriorly to innervate the triceps muscle.

F I G U R E 1 Photograph demonstrating surgical exposure of the


right side rabbit brachial plexus. Cranial is at the top of the
photograph and caudal is at the bottom. The clavicle has been divided
from its sternal attachment and reflected laterally and caudally. The
jugular vein (J) joins the subclavian vein (SV) near the origin of the
lower trunk (LT) formed by C8 and T1, obscuring their nerve roots.
The lower trunk lies between the subclavian vein and subclavian
artery (SA). C6 is usually encountered first during a supraclavicular
approach and is easily identified by its branches (**) contributing to
form the middle trunk with C7 FIGURE 3 Artists' simplified drawing of the rabbit brachial plexus

F I G U R E 2 Photograph (A) and x-ray (B) of wires inserted from nerve root through the neural foramina to identify the nerve roots
contributing to the rabbit brachial plexus. C5, C6, C7, C8, and T1 were observed to contribute significantly to the rabbit brachial plexus
4 KOLLITZ ET AL.

The rabbit triceps had three heads: long, lateral, and medial. The
medial head was short and located beneath the long and lateral heads
along the humerus. The long head originated from the scapula while
the lateral head originates from the humerus more proximally. All
three muscle bellies joined together to form a tendinous insertion
onto the olecranon.
In the electrophysiology portion of the study, four rabbits were
used to define the innervation of the triceps muscle, and three were
used to define biceps innervation.
The triceps received innervation exclusively from the radial nerve
(C6, C7, C8, and T1), which was formed primarily by the lower trunk
(C8 and T1, 12.2 mA 95% CI 81–16.3 mA) and C7 (1.9 mA, 95% CI
0.7–3.2 mA), with smaller contribution from C6 (1.6 mA 95% CI
0.4–2.8 mA) (Table 1). When connections between C7 and the lower
trunk (C8 and T1) to the triceps were severed, stimulation of C6 and
C7 no longer elicited any triceps response. When C7 was left intact
and the lower trunk was severed just distal to the union of C8 and T1,
C7 still elicited a response from the triceps muscle.
F I G U R E 4 Photograph of the rabbit brachial plexus demonstrating
The biceps was innervated proximally by a branch from C7, a
the relationship between nerve roots C5, C6, C7, C8, and T1 and upper
trunk (UT), middle trunk (MT), and lower trunk (LT). Vasculature has musculocutaneous nerve homolog, and distally by a branch from the
been removed in order to better show the nerve roots median nerve, thus every root contributed to biceps (C5–T1). Biceps
innervation was dominated by C6 (3.5 mA, 95% CI 0–7 mA) and C7
(5.0 mA, 95% CI 2.2–7.7 mA), with smaller contributions from the
lower trunk (C8 and T1, 0.9 mA, 95% CI 0.1–1.7 mA) and C5 (0.4 mA,
95%CI 0–1.1) (Table 1). The musculocutaneous homolog was primarily
derived from C6 and C7, while the branch from the median nerve
received innervation from C7 and the lower trunk via the median
nerve. When the nerve to biceps was severed, stimulation of the small
branch from the median nerve still produced a CMAP signal in the
biceps muscle. When the median nerve branch was severed, stimula-
tion of the nerve to biceps produced CMAP signal, providing electro-
physiologic evidence of a dual innervation, dominated by the
musculocutaneous homolog.

4 | DISCUSSION

The rabbit brachial plexus has a different structure as compared to the


human brachial plexus. Most investigators have used visual observational
data to identify the muscles innervated by the brachial plexus (Kawai
et al., 1989; Mohiuddin et al., 2011; Reichert et al., 2014, 2015). Reichert
F I G U R E 5 Photograph demonstrates the two nerves entering the et al. (2014, 2015) have previously described the gross morphology of
rabbit biceps muscle. The proximal tendon (A) has been released from the brachial plexus as well as nerve diameters and histomorphometry.
its intra-articular insertion. The proximal innervation (B) is derived Our study has built on this knowledge by identifying the biceps and tri-
from the musculocutaneous nerve (C6, C7) and the distal innervation ceps muscle as amenable to motor functional outcomes measurements
(C) is a branch from the median nerve (C7, C8, and T1)
and testing with isometric tetanic force, and further described the surgi-
cal approach and verified dominant innervation patterns through electro-
The rabbit biceps had only one origin, homologous to the human physiologic testing, further laying the groundwork for a surgical model of
long head. The rabbit biceps originated within the shoulder joint from brachial plexus nerve injury and repair.
the glenoid as a long tendon, and inserted broadly on the radius. We found that the biceps receives innervation primarily from C7,
Nerves were observed penetrating the muscle belly roughly 1 cm dis- with lesser contributions from C6. We also identified two sites of
tal to the musculotendinous junction proximally and distally ~1 cm innervation of the biceps, one proximal from C6 and C7, and one dis-
from the insertion. tal from a branch off the median nerve (C6, C7, and C8). The triceps
KOLLITZ ET AL. 5

TABLE 1 Electrophysiologic data

Average biceps 95% confidence Average triceps


Nerve root CMAP response (mA) interval (mA) CMAP response (mA) 95% confidence interval
C5 0.4 0–1.1 0 0
C6 3.5 0–7.0 1.6 0.4–2.8
C7 5.0 2.2–7.7 1.9 0.7–3.2
Lower trunk 0.9 0.1–1.7 12.2 8.1–16.3

Abbreviation: CMAP, compound muscle action potential.

TABLE 2 Rabbit brachial plexus and description of the biceps or triceps innervation

Publication Type of study Biceps innervation Triceps innervation


Mohiuddin et al. (2011) Anatomic observation Musculocutaneous nerve via Radial nerve via caudal
caudal trunk (C7,C8, T1, and T2) trunk (C7, C8, T1, and T2)
Reichert et al. (2014) Anatomic observation Musculocutaneous nerve (C6, C7) Radial nerve (C7, C8, and T1)
Kawai et al. (1989) Experimental—Root avulsion model Musculocutaneous nerve, as a branch Radial nerve, via middle
from median nerve formed by middle and lower portion of
and lower part of the plexus the brachial plexus
Stamatoukou et al. (2006) Experimental—C6 nerve root avulsion Musculocutaneous nerve (C6) n/a
Cao et al. (2003) Experimental—C5 and C6 root avulsion C5 and C6 n/a
Kollitz, Giusti, Friedrich, Experimental—Isometric tetanic C6 and C7 to middle trunk n/a
Bishop, and Shin (2018) force of biceps to musculocutaneous
Mencalha, Sousa, and Anatomic observation C7 and C8 to musculocutaneous, C8 and T1 to radial
Abidu-Figueiredo (2016) which gives a branch to median

received innervation from C7, C8, and T1. Previous studies have electrophysiological description of contributing nerve roots and
divided the rabbit brachial plexus into a cranial trunk consisting of C5 description of surgical landmarks will allow others to more easily repli-
and C6 nerve roots, and the caudal trunk consisting of C7, C8, and T1 cate and build upon data gathered using this model.
nerve roots (Mohiuddin et al., 2011; Reichert et al., 2014). Visual The strengths of the current investigation are therefore twofold.
inspection revealed that C6 contributes to C7, which forms a robust First, we aim to describe anatomic landmarks and surgical anatomy in
trunk with many branches. We therefore suggest an alternative termi- enough detail so as to enable other investigators to design and con-
nology be considered as follows: upper trunk consisting of C5 and C6, duct experiments with motor function as an outcome. Drawings were

the middle trunk consisting of contributions from C6 and C7, and the produced in addition to dissection photographs. The surgical approach

lower trunk consisting of C8 and T1 (Figure 4). is described in detail. Second, we use electrophysiology to verify
visual observational data. Many investigators will use electrophysiol-
Some studies confirm the observations made in this study, while
ogy in addition to observational data, and its inclusion in this study is
others conflict in the innervations and anatomy reported, and still
others do not describe the anatomy in detail. Table 2 outlines several useful for experimental design using motor function as a primary out-
come. Isometric tetanic force measurements have been described for
studies involving the rabbit brachial plexus and description of the
the rabbit biceps muscle (Kollitz et al., 2018).
biceps or triceps innervation. While Cao et al. attribute biceps root
level innervation to C5 and C6, Mencalha and Mohiuddin do not We acknowledge several limitations in this study. First, we have
narrowly focused on the innervation of the biceps and triceps and did
report involvement of C5 or C6 nerve roots. Mencalha attributes
not explore the rest of the brachial plexus. We also did not take mea-
biceps innervation to C7 and C8, while Mohiuddin attributes C7, C8,
T1, and T2 (Mencalha et al., 2016; Mohiuddin et al., 2011). Reichert surements of length or diameter of nerves, or perform his-

et al and Kollitz et al attribute biceps innervation to C6 and C7 while tomorphometry, as Reichert et al. performed these measurements
previously (Reichert et al., 2014). Second, each of the methods used to
Stamatoukou et al. report only C6 (Kollitz et al., 2018; Stamatoukou
et al., 2006). There appears to be more agreement that C8 and T1 are describe the plexus has some drawbacks.

responsible for triceps innervation. Mohiuddin and Reichert both Observation cannot distinguish between sensory and motor nerves,
and similarly cannot trace nerves back to their roots. Observation alone,
report C7 involvement, while Mencalha did not. The triceps was not
therefore, cannot determine with precision the innervation of a muscle
found as an experimental model in our literature search.
The existing literature lacks anatomic detail needed for surgical and the route those nerves travel through the plexus to arrive at the mus-
cle. Electrical stimulation is better suited to identifying the path from root
planning of experiments. Studies can be difficult to interpret given the
to terminal branch that a nerve travels. Each stimulation will cause action
differing or vague anatomical structures described. Our hope is that a
clear description of the biceps and triceps innervation along with potentials to travel both antegrade and retrograde along the axons
6 KOLLITZ ET AL.

stimulated, however. In the brachial plexus, some confusion may be cre- Kim, H. M., Galatz, L. M., Patel, N., Das, R., & Thomopoulos, S. (2009).
ated through either retrograde transmission of signal or signal jump to Recovery potential after postnatal shoulder paralysis. An animal model
of neonatal brachial plexus palsy. The Journal of Bone and Joint Surgery.
neighboring axons. While electrophysiology provides good evidence to
American Volume, 91, 879–891.
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Despite these limitations, we believe that by combining methods for Validation of isometric tetanic force as a measure of muscle recovery
describing the innervation of the biceps and triceps, we mitigate the risk after nerve injury in the rabbit biceps. Journal of Hand Surgery, 43,
488.31–488.e8.
of errors introduced by each technique. The complex branching and
Kostopoulos, E., Konofaos, P., Frazer, M., & Terzis, J. K. (2010).
crossing innervations in the rabbit brachial plexus adequately recapitulate Tubulization techniques in brachial plexus surgery in an animal model
the complex structure of the brachial plexus. The basic structure can be for long-nerve defects (40 mm). Annals of Plastic Surgery, 64, 1.
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Williams, J. P. (2015). A new animal model of brachial plexus neuralgia
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produced by injection of cobra venom into the lower trunk in the rat.
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trunk, and C8 and T1 unite to form the lower trunk. In Part 2 of this Mencalha, R., Sousa, C. A. S., II, & Abidu-Figueiredo, M., IV. (2016). Ultra-
investigation, we will apply the current description of the brachial plexus sound and gross anatomy of the brachial plexus and major nerves of
the forelimb. An anesthetic approach using the domestic rabbit
experimentally. We will induce a nerve injury at the middle trunk (C6,
(Oyctolagus cuniculus) as an experimental model 1. Acta Cirúrgica
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Reichert, P., Kiełbowicz, Z., DziĘgiel, P., Puła, B., Kuryszko, J., Gosk, J., &
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dation for Surgery of the Hand and by internal funding from the repair surgery-Histomorphometric analysis. The Anatomical Record, 298,
authors' institution. The authors have no affiliations or conflicts 444–454.
Reichert, P., Rutowski, R., Kiełbowicz, Z., Kuryszko, J., Kiełbowicz, M.,
related to the work presented in this research.
Michalak, Ł., & Bochen ska, A. (2014). The rabbit brachial plexus as an
experimental model – Anatomy and surgical approach. Polish Journal of
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AUTHOR CONTRIBUTIONS
Zoubos, A. B., & Soucacos, P. N. (2006). Phrenic nerve neurotization of
the musculocutaneous nerve with end-to-side neurorrhaphy: A short
K.M.K., G.G., P.F.F., A.T.B., and A.Y.S. contributed to the research
report in a rabbit model. Microsurgery, 26, 268–272.
design and or acquisition of data. K.M.K., G.G., P.F.F., A.T.B., Wang, H., Spinner, R. J., & Windebank, A. J. (2009). Quantitative evalua-
A.Y.S. contributed in drafting and critical revision of manuscript. tion of movement and strength of the upper limb after transection of
K.M.K., G.G., P.F.F., A.T.B., A.Y.S. contributed in approval of the final the C-7 nerve: Is it possible in an animal model? Journal of Neurosur-
gery. Spine, 10, 102–110.
submitted version.
Yang, J., Li, X., Hou, Y., Yang, Y., Qin, B., Fu, G., … Gu, L. (2015). Develop-
ment of a novel experimental rat model for brachial plexus avulsion
injury. Neuroreport, 26, 501–509.
ORCID
Yang, W., Yang, J., Yu, C., & Gu, Y. (2014). End-to-side neurotization with
different donor nerves for treating brachial plexus injury: An experi-
Patricia F. Friedrich https://orcid.org/0000-0002-6143-9784
mental study in a rat model. Muscle & Nerve, 50, 67–72.
Alexander Y. Shin https://orcid.org/0000-0001-9658-8192 Zhang, Z., Johnson, E. O., Vekris, M. D., Zoubos, A. B., Bo, J., Beris, A. E., &
Soucacos, P. N. (2006). Repair of the main nerve trunk of the upper
limb with end-to-side neurorrhaphy: An experimental study in rabbits.
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Reconstructive Microsurgery, 19, 265–270.
Ibrahim, A. G., Raisman, G., & Li, Y. (2009). Permanent loss of fore-paw How to cite this article: Kollitz KM, Giusti G, Friedrich PF,
grasping requires complete deprivation of afferent input from a mini- Bishop AT, Shin AY. The rabbit brachial plexus as a model for
mum of four dorsal roots of the rat brachial plexus. Experimental Neu- nerve injury and repair Part 1: Anatomic study of the biceps
rology, 215, 142–145.
and triceps innervation. Microsurgery. 2019;1–6. https://doi.
Kawai, H., Ohta, I., Masatomi, T., Kawabata, H., Masada, K., & Ono, K.
(1989). Stretching of the brachial plexus in rabbits. Acta Orthopaedica org/10.1002/micr.30482
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