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Micr 30482
Micr 30482
Micr 30482
DOI: 10.1002/micr.30482
RESEARCH ARTICLE
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.
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.
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 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
TABLE 2 Rabbit brachial plexus and description of the biceps or triceps innervation
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.
determine the “wiring” of a nerve, some signal spread may occur. Kollitz, K. M., Giusti, G., Friedrich, P. F., Bishop, A. T., & Shin, A. Y. (2018).
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.
thought of as roots which form three trunks that give off terminal bra- Liu, C., Qian, X., JianXiong, A., Wang, Y., Fang, Q., Jiang, Y., …
Williams, J. P. (2015). A new animal model of brachial plexus neuralgia
nches. A very small branch of C5 joins with C6 to form the upper trunk.
produced by injection of cobra venom into the lower trunk in the rat.
C6 contributes a branch to C7 which can be thought of as the middle Pain Medicine, 16, 1680–1689.
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
C7) level and study its effects on the rabbit biceps muscle, using CMAP,
Brasileira, 31, 227–234.
isometric tetanic force, and muscle weight as primary outcomes. Mohiuddin, M., Rahman, M., Alim, M., Kabir, M., & Kashem, M. A. (2011).
Macro anatomical investigation of brachial plexus of the White
New Zealand rabbit (Orycotolagus cuniculus). International Journal of
ACKNOWLEDGMENTS Natural Sciences, 1(3), 74–76.
Reichert, P., Kiełbowicz, Z., DziĘgiel, P., Puła, B., Kuryszko, J., Gosk, J., &
This work was supported in part by a grant from the American Foun- Bochen ska, A. (2015). The rabbit brachial plexus as a model for nerve
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
Veterinary Sciences, 17, 339–345.
CONF LICT OF IN TE RE ST
Riva, N., Domi, T., Lopez, I. D., Triolo, D., Fossaghi, A., Dina, G., …
Quattrini, A. (2012). The brachial plexus branches to the pectoral mus-
No author has a conflict of interest regarding this paper.
cles in adult rats: Morphological aspects and morphometric normative
data. Frontiers in Neuroanatomy, 6, 41.
Stamatoukou, A., Papadogeorgou, E., Zhang, Z., Pavlakis, K.,
AUTHOR CONTRIBUTIONS
Zoubos, A. B., & Soucacos, P. N. (2006). Phrenic nerve neurotization of
the musculocutaneous nerve with end-to-side neurorrhaphy: A short
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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-
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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
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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
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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
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