INTRODUCTION
The three-toed sloths comprise four species: Bradypus variegatus Schinz, 1825, with a wide distribution from Central America to South America; Bradypus tridactylus Linnaeus, 1758, occurring only in the Amazon region of Brazil, Venezuela, Bolivia and Guyana; and Bradypus torquatus Illiger, 1811, endemic to the Atlantic Forest on the Brazilian southeast coast. The latter species was distributed from northern Rio de Janeiro to Pernambuco, but currently survives only in Atlantic Forest corridors in southeastern Bahia, near Ilhéus, southern Espírito Santo and the outskirts of the city of Rio de Janeiro. The species most recently described, Bradypus pygmaeusAnderson & Handley (2001) is currently considered to be at critical risk of extinction, with a population restricted to a small island on the Caribbean coast of Panama (Wetzel 1985; Fonseca et al. 1996; Aguiar & Fonseca 2008).
Bradypus variegatus Schinz, 1825, is commonly known as common sloth or marloth sloth or brownthroat sloth (Superina & Aguiar 2006). This species occurs from Honduras, west of the coast of Ecuador, Colombia and Venezuela, east of the Andes and some forests of Ecuador, Peru, Bolivia and Brazil, with the exception of the Amapá and north of the Pará (Wetzel 1982). The brachial plexus is a complex anatomical structure formed by a varied set of joints between the ventral branches of the last cervical and thoracic spinal nerves (Martini et al. 2009).
Ecomorphological studies allow to deduce how species interact with their habitat, and may even be used in favor of conservation (Moura et al. 2007). In this context, the comparative anatomy of brachial plexus formation has been studied since the nineteenth century (Paterson 1887) and remains one of the most current issues of contemporary research (Johnson et al. 2010). The levels of origin of the brachial plexus may stem from genetic and embryonic factors, represented by the position in which the limb buds develop in relation to the neuro-axis (Parada et al. 1989). Information on the comparative anatomy of the brachial plexus may verify the hypothesis that in the evolutionary process, cranial migration occurred of the spinal nerve branches that form the plexus throughout the evolutionary history (Parada et al. 1989). The morphological descriptions of the brachial plexus in Bradypus variegatus are scarce. Currently these animals can be found in nurseries and reserves, requiring high qualification and training of professionals working in this area.
Therefore, increased information generated on the anatomy of this species will support clinical-surgical approaches and certainly contribute to the preservation and well-being of these animals.
The objectives of this research were to determine the origin and antimeric distribution of the brachial plexus nerves in Bradypus variegatus, their anatomical variations, and to establish morphological parameters of the respective nerves, contributing information for the comparative anatomy of wild animals.
MATERIAL AND METHODS
The Ethics and Research Committee of the Federal Rural University of Rio de Janeiro approved this research (CEUA No. 018/2017).
Twelve adult sloth (Bradypus variegatus Schinz, 1825) of both sexes (six males and six females) were used. These came from the Serra dos Órgãos National Park and the Wild Animals Rehabilitation Center of Estácio de Sá University, with different history of deaths from natural causes, and donated to the Department of Animal and Human Anatomy of the Federal Rural University of Rio de Janeiro. For this research, two thoracic limbs of each animal were dissected. The animals were previously identified and fixed with perfusion of 10% formalin aqueous solution using the common carotid artery accessed by ventral median cervical incision, in addition to intramuscular infusions and in the body cavities. After the procedures described, the specimens were packed in 500 l, low-density polyethylene boxes containing formaldehyde solution at the same concentration for a period of approximately 2 months.
After completing this period, the animals were washed in running water and submitted to radiographic examinations of the cervical region (Fig. 1) in the Imaging Department of the Small Animal Veterinary Hospital of the Federal Rural University of Rio de Janeiro, in order to characterize the number of cervical vertebrae. Radiographs were obtained at the ventro-dorsal and laterolateral positions. Using basic dissecting materials, the thoracic viscera were removed, as well as the adipose tissue of the region, partially exposing the nerves from the plexus under study. Such dissection was performed under a mesoscopy technique, first removing the sternum and thoracic viscera and then the vertebral bodies until the spinal cord and the roots forming the plexus were completely exposed, bein possible to analyze the origin and composition of the nerves from the brachial plexus.
To better visualize the nerves, imbibition process used absolute alcohol solution and acetic acid in the ratio of 70/30 respectively, for 30 to 60 minutes, as described by (Ribeiro et al. 2005).
The Veterinary Anatomic Nomenclature was used (ICVGAN 2017). The photographic records of all the plexus were made with digital camera (Nikon D7200 24.2 MP).
The results of the origin and antimeric distribution of the nerves were expressed in absolute frequency and simple percent frequency.
RESULTS
The dissections allowed to register and characterizethe origin and antimeric distribution of the nerves that make up the brachial plexus of the 12 Bradypus variegatus specimens. The radiographic examination (Fig. 1) revealed nine cervical vertebrae in all studied specimens.
Origin of the brachial plexus Brachial plexuses were the result of the junctions between the ventral branches of the last three spinal nerves (C8, C9, C10) and the first and second thoracic nerves (T1 and T2). The right and left phrenic nerves (belonging to the cervical plexus) originated from C8 and C9, though they did not participate in the brachial plexus. There was an antimeric variation in the origin in some individuals.
After analyzing the total of 13 nerves dissected in the 24 plexus (n = 312), it was observed that the ventral spinal branches that contributed most to the formation of the nerves were C8 (55.4%), followed by C9 (54.5%), C10 (41.0%), T1 and T2 (40.4%). The total contributions to the formation of the plexus were similar between the antimeres: 359 in the right and 363 in the left. It was observed that the ventral branches C8 and C9 had a greater antimeric contribution than C10, T1 and T2. The 722 dissected ventral branches formed 312 nerves. Thus, each nerve was formed on average by 2.3 branches. The brachial plexus derived the suprascapular, subscapular, radial,axillary, medial, ulnar and musculocutaneous nervesto innervate the intrinsic musculature (Table 1).
The nerves of the cranial pectoral, caudal pectoral, lateral thoracic, thoracic, thoracodorsal and tensor muscles of the forearm supply the extrinsic muscles (Table 2).
The conformation of the ventral branches of the plexus showed a variable form in the right and left antimeres (Fig. 2).
Nervous trunks
In all specimens, the ventral branches formed two nerve trunks and two fascicles (Fig. 3): cranial trunk and caudal trunk, dorsal fasciculus, and ventral fasciculus. After the trunks are formed, the union occurs resulting in a common trunk. From this point on, dorsal and ventral fascicles are observed in antimeres of both of specimen’s (Fig. 4).
The cranial trunk originated from the union of the ventral branches of C8 and C9, with the participation of C10 in some cases. From the cranial trunk, the suprascapular and subscapular nerves formed obligatory contributions to the axillary, musculocutaneous, radial, medial, musculocutaneous, thoracic lateral and thoracodorsal nerves, and eventually to the long thoracic and cranial pectoral nerves (33.3%), caudal pectoral (25%).
The caudal trunk was formed by the ventral branches of C10, T1 and T2. The same contributed to the origin of the C10 subscapular nerves (five right and five left antimeres - 41.7%), radial, axillary at C10 (six right and eight left antimeres - 50% and 66.7%, respectively), median, ulnar, musculocutaneous, cranial pectoral, caudal pectoral, thoraciclateral and long thoracic in C10 (a left antimere -8.3%), thoracodorsal and tensor of the fascia of theforearm.
The axillary, thoracodorsal, lateral thoracic, thoracic, cranial, radial, suprascapular and subscapular nerves had common origin in the dorsal fascicle. The ventral fascicles originated the musculocutaneous nerves (considered a nerve in part, despite the origin common to the median nerve); median, caudal, ulnar and forearm fascia tensor originated from the ventral fascicle.
The ulnar nerve traversed the arm without issuing branches, going to the forearm. The median and musculocutaneous nerves followed the medial side of the arm as a common trunk. In its middle third, the musculocutaneous nerve was detached to innervate the coracobrachial and biceps muscles. The median nerve attached to the musculocutaneous nerve traversed the foramen with the brachial artery and proceeded to the forearm.
An outline of the spinal cord, spinal and nerves of the right plexus is shown in Fig. 5.
DISCUSSION
Origin of the brachial plexus
A total of 24 dissected plexuses of Bradypus variegatus were derived from the connections between the ventral branches of the last three spinal nerves (C8, C9, C10) and the first and second thoracic nerves (T1 and T2). This formation, based on the last three cervical spinal segments and the first two thoracic segments, resembles in part the results obtained in different primate species such as Saimiri sciureus (Araújo et al. 2012), Galago senegalensis (Kanagasuntheram & Mahran 1960), Papio ursinus (Booth et al. 1997), Lagothrix lagothricha (Cruz & Adami 2010) and Macaca mulatta (Lu et al. 2013) and in man (Zhang et al. 2016; Guday et al. 2017).
Mammals of other orders having the plexus formed by at least five ventral branches include monotremes (Koizumi & Sakai 1997), Myocastor coypus (Guimarães et al. 2013), Bradypus variegatus (Amorim Júnior et al. 2003), Hydrochoeris hydrochaeris (Fioretto et al. 2003), Agouti paca (Scavone et al. 2008), Tamandua tetradactyla (Cruz et al. 2012), Myrmecophaga tridactyla (Souza et al. 2014), Leopardus geoffroyyi (Souza et al. 2017) and human and nonhuman primates in general (Booth et al. 1997; Cruz & Adami 2010; Zhang et al. 2016; Guday et al. 2017).
There was no participation of C4, C5, C6 and C7 in any dissected animal, different from that described by Amorim Júnior et al. (2003) for Bradypus variegatus. Perhaps this difference can be attributed, in part, to the counting of the cervical vertebrae. In the present study, accurate counting was made possible by radiographic exploration. Carpenter (1978) and Moura et al. (2007) suggest that changes in the origin of the plexus are due to variations in the insertion position of the limbs in relation to the neuro-axis. This argument seems to be more reasonable, since the origin in more cranial branches is not unique for more derived species, since the presence of C4 in the brachial plexus formation was also described for monotremes (Koizumi & Sakai 1997), Bradypus variegatus (Amorim Júnior et al. 2003), Hydrochoerus hydrochaeris (Fioretto et al. 2003) and Pecari tajacu (Moura et al. 2007).
As proposed by (Allam et al. 1952), the plexus formed by only four ventral branches are characters of species whose thoracic limbs are limited to the support of specialized body mass and locomotion and constitutively devoid of clavicle, such as canids (Souza et al. 2014; 2017) and ungulates (Getty et al. 1986). Thus, the presence of at least five ventral branches in the B. variegatus brachial plexus may be a reflection of a more versatile performance of the thoracic limb when compared with specialized cursors.
The contribution of T2, observed in Bradypus variegatus, was verified in Chinchilla lanigera (Cevik Mirkan et al. 2007), Hystrix cristata (Aydin 2003) and humans (Johnson et al. 2010; Guday et al. 2017). This type of participation constitutes “post-fixed” (absence of the ventral branch C4 in the brachial plexus formation and presence of the ventral branch T2) type plexuses (Parada et al. 1989). In the case of post-fixed plexus in humans, the lower trunk (caudal) can be compressed by the first rib and induce neurovascular changes in the thoracic limb (Guday et al. 2017).
The brachial plexus of Bradypus torquatus originated from the spinal nerves C7 to C10 and T1 to T2, with the participation of T2 variable (Cruz et al. 2013), different from the origin observed herein which the brachial plexus had as its cranial origin the C8 branch and invariably T2 as the most caudal origin.
Studies that account for the branches that contributed most to the formation of the plexus are still restricted. In the specimens of B. variegatus, we observed that C8 and C9 branches contributed most to the formation of nerves. Souza et al. (2014; 2017) observed in Cerdocyon thous and Lycalopex gymnocercus that the roots that contributed most to the formation of nerves came possibly from C7 or C8. This difference is because the origins of the plexus described in carnivores, as well as the position of the thoracic limbs in relation to the neuro-axis, are slightly caudally oriented when compared with those described in other mammals. However, no other species were found in which the most cranial spinal branch contributes most to the formation of the plexus, as foccurs in Bradypus variegatus.
Nervous Trunks
In all the sloths of the Bradypus variegatus species dissected in the present investigation, the ventral branches formed two nerve trunks: cranial trunk and caudal trunk.
The cranial trunk originated from the union of the roots of C8 and C9. The caudal trunk was formed through the ventral roots of T1 and T2. In both logs, there was a variable participation of C10.
Trunk formation is commonly described in humans (Johnson et al. 2010) and in nonhuman primates (Cruz & Adami 2010; Kikushi et al. 2011). Nonprimate mammals, such as monotremes (Koizumi & Sakai 1997), Hystrix cristata (Aydin 2003), Sciurus vulgaris (Aydin 2011), Hippopotamus amphibius (Yoshitomi et al. 2012), Tamandua tetradactyla (Cruz et al. 2012) and Bradypus torquatus (Cruz et al. 2013), also form trunks in the constitution of the plexus. However, trunks are not identified in several otherspecies of mammals, being absent in domestic mammals (Getty et al. 1986).
However, Araújo et al. (2012) reported for Saimiri sciureus, the ventral spinal branches forming four trunks, the first (cranial) formed by the spinal nerves of C4, the second (mid-cranial) by C5 and C6, the third (medium-flow) by C7 and C8, and the fourth (flow rate) originated in T1.
In Bradypus torquatus, the spinal nerves originated in the cranial and caudal trunks which united and formed a common trunk that emitted two fascicles, from which all nerves of the brachial plexus originated, with the exception of the pectoral nerves, thoracic long and scapular, which emerged before the formation of the common trunk (Cruz et al. 2013), corroborating in part with the results obtained in the present study. Aydin (2003) reports that in the brachial plexus of Hystrix cristata, the ventral branch of C5 and the ventral branch of C6 formed the cranial trunk, and cranial branch of T2 and ventral branches of C7, C8 and T1 formed the caudal trunk, which is the biggest.
Similar to research carried out by other authors, Cruz et al. (2012) found in the brachial plexus of Tamandua tetradactyla that the spinal nerves originated in three trunks: cranial, middle and caudal. In this case, C5 and C6, C7, C8 and T1, respectively. The origin of the brachial plexus of Myocastor coypus is composed of the segmental branches of the spinal cord, the intervertebral spaces of the cervical vertebrae (C6, C7 and C8) and thoracic vertebra (T1), originating three main trunks (Guimarães et al. 2013). The plexus of B. torquatus consisted mainly of two trunks and two fascicles that derived the nerves, also to the one observed in Bradypus tridactylus (Bielek 1937). Cruz & Adami (2010) when describing the origin of the brachial plexus in Tamandua tetradactyla, a species of the Xenarthra Superorder, in which Bradypus torquatus is also found, described the origin of the brachial plexus from three trunks, different from the two trunks observed in Bradypus variegatus and also in Hystrix cristata (Aydin 2003). Nerves resulting (antimeric distribution) The suprascapular nerve was derived mostly from the first two branches that form the brachial plexus (C8 and C9) in 87.5% of the sample of Bradypus variegatus. Less frequently, this nerve originated exclusively by C8 (12.5%). These findings resemble in part those described for Tamandua tetradactyla (Cruz et al. 2012), Cerdocyon thous (Souza et al. 2014), Priodontes maximus (Fernandes et al. 2015), Macaca mulatta (Santos-Sousa et al. 2016) and Lycalopex gymnocercus (Souza et al. 2017), which although they have a more cranial origin, were also originated by the first two cervical spinal branches that make up the brachial plexus. Cruz et al. (2013) reported for Bradypus torquatus, the suprascapular nerve was also mostly originated by the first two branches of the brachial plexus. However, these authors report that the formation of the brachial plexus in B. torquatus has its origin in C7. Therefore, for these authors, the suprascapular nerve was originated mostly by C7 and C8 and supplied the supraspinatus, infraspine and deltoid muscles, resembling in parts our observations. However, innervation of the deltoid muscle was not observed in our analyses.
The subscapular nerve in Bradypus variegates was formed by branches C8 and C9 in 50% of the sample, although C10 also contributed to its formation. As described by Cruz et al. (2013) for B. torquatus, subscapular nerves were in varied numbers of 3 to 4 branches, which supplied exclusively the subscapularis muscle. Variations in the numbers of subscapular nerves are commonly reported in primates such as Gorilla sp., Pan troglodytes and Pongo sp. (Hepburn 1891), C. apella (Ribeiro et al. 2005) and M. mulatta (Santos-Sousa et al. 2016).
The radial nerve was formed by the contribution of all branches of the brachial plexus in 100% of the sample. These findings are in agreement with those described by Booth (1991) for Papio ursinus. The radial nerve delivered branches still on the medial face to the portions of the brachial triceps, then circumvented the humeral diaphysis appearing on the lateral face after issuing branches to the anconeous muscle continuing distally to the muscles of the forearm and hand. Similar results were observed by Cruz & Adami (2010) in a study with Lagothix lagotricha; although the authors did not find innervation for the anconeous muscle. The territory of innervation described here resembles those described by Araújo et al. (2012) for S. sciureus, Cruz et al. (2013) for B. torquatus and Santos-Sousa et al. (2016) for Macaca mulatta.
The axillary nerve was most frequently originated by the contributions of branches C8, C9 and C10, similar to described by Hepburn (1891) in anthropoid primates, where the formation of this nerve occurs by the contributions of the first three branches of the brachial plexus. In its path, the axillary nerve innervated the muscles larger round (teres major), smaller round (teres minor) and deltoid. Such results were also described for Lagothrix lagotricha (Cruz & Adami 2010), Saimiri sciureus (Araújo et al. 2012) and Macaca mulatta (Santos-Sousa et al. 2016). However, Cruz et al. (2012) did not observe innervation for the smaller round muscle (teres minor) of Bradypus variegatus; founding a branch for an accessory portion of the biceps brachii, differing in part with our observations.
The median nerve formed from the ventral roots of C8 to T2 in 91.6% of the sample, and only in an antimere was the exclusive participation of C8 and C9. According to Santos-Sousa et al. (2016), there is consensus among authors that the median nerve has a broad contribution of the different spinal branches as verified in our analyses. In addition, a particularity founded here was the formation of musculocutaneous nerve from the median nerve, in which they formed a single trunk in almost all its course, which is in agreement with the reported by Atoji et al. (1987) for Japanese antelopes; Fioretto et al. (2003) for Hydrochoerus hydrochaeris and Cruz et al. (2013) for Bradypus torquatus. The formation of a single trunk between the brachial plexus nerves, especially between the median and ulnar nerves, has been reported for domestic carnivores (Getty et al. 1986), Arctocephalus australis (Souza et al. 2010) and Cerdocyon thous (Souza et al. 2014) and, recently for Lycalopex gymnocercus (Souza et al. 2017).
The ulnar nerve in 95.8% of the sample originated from the T1 and T2 branches and followed medially without emitting branches to arm muscles. In the distal epiphysis of the humerus, the nerve is directed to the forearm innervating the flexor muscles of the carpus, bypassing the medial epicondyle, which is in agreement with the observed for mammals in general (Cruz et al. 2013).
The musculocutaneous nerve in Bradypus variegatus has the same origin as the median nerve. The musculocutaneous nerve is derived from a common part, called the median nerve musculocutaneous (Atoji et al. 1987; Fioretto et al. 2003; Cruz et al. 2013). It supplied the coracobrachial, brachial and biceps brachii muscles, as observed by Cruz et al. (2013) for B. torquatus.
The cranial pectoral nerves were invariably formed by branches C8 and C9, with varying contributions of C10. This more cranial origin resembles in part the findings described by Santos-Sousa et al. (2016) for M. mulatta and differ from those described by Cruz et al. (2013) for Bradypus. However, it is worth mentioning that these authors did not distinguish the cranial and caudal pectoral nerves, grouping them into the same nomenclature (NnPpectoral nerves). Nevertheless, even for these authors, the described origin of these nerves came from the caudal trunk.
Research involving the anatomy of the brachial plexus and, consequently, the description of the pectoral nerves does not present a homogeneous terminology. Ribeiro et al. (2005) in a study with Cebus apella and Cruz & Adami (2010) in Lagothrix Lagotricha described minor and major pectoral nerves. It is important to note that the cranial nerve palsies are those that supply the superficial pectoralis muscle, and therefore the pectoral nerves are those that supply the deep pectoral muscle (ICVGAN 2017).
The caudal nerve was completely formed by branches of T1 and T2 with varied contributions of C10. These findings resemble those described in Bradypus torquatus (Cruz et al. 2013) and M. mulatta (Santos-Sousa et al. 2016).
With its origin starting from the pectoral nerves (cranial and caudal, variably), the lateral thoracic nerve was originated most frequently by the contributions of C8, C9 and C10, that naturally contribute to the formation of the pectoral nerves. The lateral thoracic nerve innervated part of the deep pectoral muscle and mainly the cutaneous musculature of the trunk. These findings resemble those described for domestic dog (Evans & De Lahunta 2013), Arctocephalus australis (Souza et al. 2010), Cerdocyon thous (Souza et al. 2014) and Lycalopex gymnocercus (Souza et al. 2017). However, for Bradypus, this nerve was not reported (Cruz et al. 2013).
The long thoracic nerve was recorded in just one antimere in two specimens. Its reported absence in our results may have been encouraged by the small nerve diameter and fixation limitations. In these records, it originated in (C10) and innervated the ventral serratus muscle, resembling in part the results of Cruz et al. (2013) for Bradypus torquatus, since the reported origin restricts the contribution of C9. The innervation of the ventral serratus muscle in Bradypus variegatus is in agreement with the observed in most mammals.
The thoracodorsal nerve, originated by the contributions of all branches that compose the brachial plexus of Bradypus variegatus, innervates the great dorsal muscle. These results are in agreement with those described by Booth et al. (1997) for Papio ursinus, because although the origin of the plexus is more cranial in the primates, it also occurred the participation of all branches forming the plexus. In Bradypus torquatus, Cruz et al. (2013), observed the emission of branches to the larger round muscle (teres major) in two animals.
CONCLUSION
There was antimeric symmetry in relation to the origin of the plexus, opposing the resulting nerves in all the animals of the present study.
Bradypus variegatus brachial plexuses are formed by the contributions of the ventral spinal branches of the last three spinal nerves (C8, C9, C10) and the first and second thoracic nerves (T1 and T2).
The branches of C8 and C9 contributed the most to the formation of brachial plexus nerves. It was possible to observe cases of absences of some branches of almost all the nerves that are distributed in the extrinsic musculature in both antimeres.
In Bradypus variegatus, the musculocutaneous nerve presented origin common to the median nerve. These nerves are formed by a common trunk called the musculocutaneous median nerve.
Due to the origin of the brachial plexus in Bradypus variegatus and participation of T2 (second thoracic spinal nerve) in its formation, it can becharacterized as post-fix.