Axonal profiling of the common fibular nerve and its branches: Their functional composition and clinical implications.

The articular branch (Arb) from the common fibular nerve (CFN) plays a pivotal role in procedures such as genicular nerve blocks since it extensively innervates the anterolateral knee joint. It remains unclear whether the Arb can be classified as purely sensory, and understanding its axonal composition is critical to prevent muscle weakness during nerve blocks. We conducted a histological analysis on six cadaveric nerve specimens (four males and two females; mean age at death, 81.3 years old). The axonal composition of the main trunk of the CFN, the deep and superficial fibular nerves (DFN and SFN), and the Arb was verified through double immunofluorescence labeling with antibodies against neurofilament 200 and choline acetyltransferase. We revealed that the DFN contains motor and sensory fascicles that serve the anterior muscular compartment of the leg, including the fibularis longus and the first web space of the foot. Moreover, we showed that the SFN includes a major sensory branch innervating the skin of the lateral leg and the dorsum of the foot and a minor motor branch for the lateral muscular compartment of the leg. Furthermore, we demonstrated that the Abr contains a major sensory branch that targets the infrapatellar fat pad, the knee joint, and a minor motor branch innervating the superior part of the anterior muscular compartment of the leg. Thus, our study proves that the Arb is a motor-sensory mixed nerve, suggesting that an Arb block may significantly weaken the anterior leg muscles.

Neuromuscular compartmentation of the subscapularis muscle and its clinical implication for botulinum neurotoxin injection.

In this study, using immunohistochemistry with fresh cadavers, deliberate histological profiling was performed to determine which fibers are predominant within each compartment. To verify the fascial compartmentation of the SSC and elucidate its histological components of type I and II fibers using macroscopic, histological observation and cadaveric simulation for providing an anatomical reference of efficient injection of the BoNT into the SSC. Seven fixed and three fresh cadavers (six males and four females; mean age, 82.5 years) were used in this study. The dissected specimens revealed a distinct fascia demarcating the SSC into the superior and inferior compartments. The Sihler's staining revealed that the upper and lower subscapular nerves (USN and LSN) innervated the SSC, with two territories distributed by each nerve, mostly corresponding to the superior and inferior compartments of the muscle, although there were some tiny communicating twigs between the USN and LSN. The immunohistochemical stain revealed the density of each type of fiber. Compared with the whole muscle area, the densities of the slow-twitch type I fibers were 22.26 ± 3.11% (mean ± SD) in the superior and 81.15 ± 0.76% in the inferior compartments, and the densities of the fast-twitch type II fiber were 77.74% ± 3.11% in the superior and 18.85 ± 0.76% in the inferior compartments. The compartments had different proportions of slow-fast muscle fibers, corresponding to the functional differences between the superior compartment as an early-onset internal rotator and the inferior compartment as a durable stabilizer of the glenohumeral joint.

Intramuscular innervation of the tensor fasciae latae: Application to total hip arthroplasty.

The aims of this study were to clarify the extra- and intramuscular branching patterns of the tensor fasciae latae (TFL) with reference to surface landmarks on the thigh and to thus suggest a safe approach for total hip arthroplasty. Sixteen fixed and four fresh cadavers were dissected and subjected to the modified Sihler's staining method to reveal the extra- and intramuscular innervation patterns, and the findings were matched with surface landmarks. The landmarks were measured from the anterior superior iliac spine (ASIS) to the patella and divided into 20 parts along the total length. The average vertical length of the TFL was 15.92 ± 1.61 cm, which was 38.79 ± 2.73% when converted to a percentage. The entry point of the superior gluteal nerve (SGN) was an average of 6.87 ± 1.26 cm (16.71 ± 2.55%) from the ASIS. In all cases, the SGN entered parts 3-5 (10.1%-25%). As the intramuscular nerve branches traveled distally, they had a tendency to innervate more deeply and inferiorly. In all cases, the main SGN branches were intramuscularly distributed in parts 4 and 5 (15.1%-25%). Most tiny SGN branches were found inferiorly in parts 6 and 7 (25.1%-35%). In three of 10 cases, very tiny SGN branches were observed in part 8 (35.1%-38.79%). We did not observe SGN branches in parts 1-3 (0%-15%). When information on the extra- and intramuscular nerve distributions was combined, we found that the nerves were concentrated in parts 3-5 (10.1%-25%). We propose that damage to the SGN can be prevented if parts 3-5 (10.1%-25%) are avoided during surgical treatment, particularly during the approach and incision.

Retrodural space of Okada in the posterior ligamentous complex region: clinical and anatomical findings relevant to lumbar interlaminar epidural injection.

BACKGROUND: The retrodural space of Okada is a potential space posterior to the ligamentum flavum that allows communication with the bilateral facet joints. However, the actual anatomy of this space has not been clearly visualized to date. We sought to investigate the characteristics of patients showing contrast spreading to the facet joint space during epidural injection and to clarify the anatomical structures of the retrodural space and adjacent ligamentous tissues in cadaveric specimens. METHODS: Fluoroscopic images of patients who underwent fluoroscopy-guided lumbar interlaminar epidural injection were assessed for contrast flow to the facet joints. Patient demographics, preprocedural imaging study findings, and epidural approaches were analyzed. The anatomical study included the sectional dissection, micro-CT imaging, and histological evaluation of lumbar spine specimens from 16 embalmed cadavers. RESULTS: Fluoroscopic images of 605 epidural injections were analyzed. Among them, 36 with inadvertent spread into the facet joints (5.9%) were identified. Multivariate analysis revealed that facet joint pathologies were significantly associated with inadvertent spread into the facet joints (OR 4.382; 95% CI 1.160 to 16.558; p=0.029). Micro-CT and histological findings consistently showed a retrodural space between the ligamentum flavum and interspinous ligament. Various anatomical communication routes in the posterior ligamentous complex leading to this space were observed in specimens with degenerative and pathological changes. CONCLUSION: Degenerative and pathological facet joint changes were associated with a higher incidence of spread into the retrodural space during epidural injection. Our findings confirm anatomical evidence for a false loss of resistance before the needle enters the epidural space.

Delineation and histological examination of the intramuscular innervation of the platysma: Application to botulinum neurotoxin injection.

This study aimed to identify the whole innervation pattern of the platysma using the Sihler's staining, and the axonal composition profile of the sensory-motor anastomosis identified by immunofluorescence assays. The findings provide a comprehensive understanding of the neural anatomy of the platysma and facilitate efficient and safe manipulation for neurotoxin injection. Ten fixed and two fresh hemifaces were included in this study. Sihler's staining was used to the study 10 fixed hemifaces and two fresh hemifaces were used for immunofluorescence assays. In all cases, the cervical branch of facial nerve (Cbr) broadly innervated the platysma, and the marginal mandibular branch of facial nerve (MMbr) provided supplementary innervation to the uppermost part of the platysma. The transverse cervical nerve (TCN), great auricular nerve (GAN), and supraclavicular nerve (SCN) were observed in the lower half of the platysma. In 30% of all cases, there was a communicating loop between the Cbr and TCN. In 20% of all the cases, a communicating branch joined between the Cbr and GAN. For successful esthetic rejuvenation procedures, a clinician should consider the Cbr distribution to the overall platysma and additionally innervation by individual nerves (MMbr, GAN, TCN, and SCN) to the middle and lower portions of the platysma muscle.

Anatomical study of the adductor canal: three-dimensional micro-computed tomography, histological, and immunofluorescence findings relevant to neural blockade.

BACKGROUND: A precise anatomical understanding of the adductor canal (AC) and its neural components is essential for discerning the action mechanism of the AC block. We therefore aimed to clarify the detailed anatomy of the AC using micro-computed tomography (micro-CT), histological evaluation, and immunofluorescence (IF) assays. METHODS: Gross dissections of 39 thighs provided morphometric data relevant to injection landmarks. Serial sectional images of the AC were defined using micro-CT and ultrasonography. The fascial and neural structures of the AC proper were histologically evaluated using Masson's trichrome and Verhoeff-Van Gieson staining, and double IF staining using choline acetyltransferase (ChAT) and neurofilament 200 antibodies. RESULTS: The posteromedial branch insertion of the nerve to vastus medialis (NVM) into the lateral border of the AC proper was lower (14.5 ± 2.4 cm [mean ± SD] above the base of the patella) than the origin of the proximal AC. The AC consists of a thin subsartorial fascia in the proximal region and a thick aponeurosis-like vastoadductor membrane in the distal region. In the proximal AC, the posteromedial branch of the NVM (pmNVM) consistently contained both sensory and motor fibers, and more ChAT-positive fibers were observed than in the saphenous nerve (27.5 ± 11.2 / 104 vs. 4.2 ± 2.6 / 104 [counts/µm2], P < 0.001). CONCLUSIONS: Anatomical differences in fascial structures between the proximal and distal AC and a mixed neural component of the neighboring pmNVM have been visualized using micro-CT images, histological evaluation, and IF assays.

An anatomical and radiological study of the tectorial membrane and its clinical implications.

The radiological image of an intact tectorial membrane (TM) became an important favorable prognostic factor for craniovertebral instability. This study visualized the fascial layers of the TM and adjacent connective tissues with clinical significance by micro-CT and histological analysis. The TM firmly attached to the bony surface of the clivus, traversed the atlantoaxial joint posteriorly, and was inserted to the body of the axis showing wide distribution on the craniovertebral junction. The supradental space between the clivus, dens of the axis, anterior atlantooccipital membrane, and the TM contained profound venous networks within the adipose tissues. At the body of the axis, the compact TM layer is gradually divided into multiple layers and the deeper TM layers reached the axis while the superficial layer continued to the posterior longitudinal ligament of the lower vertebrae. The consistent presence of the fat pad and venous plexus in the supradental space and firm stabilization of the TM on the craniovertebral junction was demonstrated by high-resolution radiologic images and histological analysis. The evaluation of the TM integrity is a promising diagnostic factor for traumatic craniovertebral dislocation.

The pathway of injectate spread during thoracic intertransverse process (ITP) block: Micro-computed tomography findings and anatomical evaluations.

STUDY OBJECTIVE: To investigate the mechanism of action of the thoracic intertransverse process (ITP) block. DESIGN: Three-dimensional micro-computed tomography (3D micro-CT) study and cadaveric evaluation. SETTING: A translational research unit for anatomy and analgesia in a university hospital. PATIENTS: Twelve embalmed and three non-embalmed human cadavers were used in this study. MEASUREMENTS: Micro-CT images of the mid-thoracic paravertebral space and its adjacent ligamentous tissues were acquired and 3D images were reconstructed. Manual dissection and histologic examination of these structures complemented the images. To confirm our findings, the dye-spreading pattern after ultrasound-guided ITP injection of 20 mL dye solution at T4-T5 was evaluated. MAIN RESULTS: Micro-CT and histologic findings showed that the costotransverse foramen (the medial slit of the superior costotransverse ligament) and the costotransverse space (between the rib and the transverse process) were potential pathways to the thoracic paravertebral space during ITP block. Single-level ITP injection with a dye solution resulted in a multilevel segmental paravertebral spread in cadaveric evaluation. CONCLUSIONS: The space posterior to the superior costotransverse ligament, the target area for ITP blocks, has potential anatomical pathways to the thoracic paravertebral space. The costotransverse foramen and the costotransverse space provided the anatomical conduit for the anterior and intersegmental paravertebral spread of the ITP block.

Micro-computed tomography with contrast enhancement: An excellent technique for soft tissue examination in humans.

Manual dissection and histologic examination are commonly used to investigate human structures, but there are limitations in the damage caused to delicate structures or the provision of limited information. Micro-computed tomography (microCT) enables a three-dimensional volume-rendered observation of the sample without destruction and deformation, but it can only visualize hard tissues in general. Therefore, contrast-enhancing agents are needed to help in visualizing soft tissue. This study aimed to introduce microCT with phosphotungstic acid preparation (PTA-microCT) by applying the method to different types of human tissue. Specimens from human cadavers were used to examine the orbicularis retaining ligament (ORL), nasolabial fold (NLF), and the calcaneal tunnel of the sole. Using PTA-microCT, relevant information of human structures was identified. In the ORL study, tiny and delicate ligamentous fibers were visualized in detail with multidirectional continuity. In the NLF study, complex structural formation consisting of various types of soft tissue were investigated comprehensively. In the calcaneal tunnel study, the space surrounded by diverse features and its inner vulnerable structures were examined without damage. Consequently, we successfully applied the PTA-microCT technique to the analysis of specific human soft tissue structures that are challenging to analyze by conventional methods.

Anatomy of the thoracic paravertebral space: 3D micro-CT findings and their clinical implications for nerve blockade.

BACKGROUND: A precise anatomical understanding of the thoracic paravertebral space (TPVS) is essential to understanding how an injection outside this space can result in paravertebral spread. Therefore, we aimed to clarify the three-dimensional (3D) structures of the TPVS and adjacent tissues using micro-CT, and investigate the potential routes for nerve blockade in this area. METHODS: Eleven embalmed cadavers were used in this study. Micro-CT images of the TPVS were acquired after phosphotungstic acid preparation at the mid-thoracic region. The TPVS was examined meticulously based on its 3D topography. RESULTS: Micro-CT images clearly showed the serial topography of the TPVS and its adjacent spaces. First, the TPVS was a very narrow space with the posterior intercostal vessels very close to the pleura. Second, the superior costotransverse ligament (SCTL) incompletely formed the posterior wall of the TPVS between the internal intercostal membrane and vertebral body. Third, the retro-SCTL space broadly communicated with the TPVS via slits, costotransverse space, intervertebral foramen, and erector spinae compartment. Fourth, the costotransverse space was intersegmentally connected to the adjacent retro-SCTL space. CONCLUSIONS: A non-destructive, multi-sectional approach using 3D micro-CT more comprehensively demonstrated the real topography of the intricate TPVS than previous cadaver studies. The posterior boundary and connectivity of the TPVS provides an anatomical rationale for the notion that paravertebral spread can be achieved with an injection outside this space.

Analysis of the positional relationship of the long thoracic nerve considering clinical treatment.

INTRODUCTION: The long thoracic nerve (LTN) has a risk of being damaged during chest surgery and should be considered when performing anesthesia such as a serratus anterior plane block (SAPB). We analyzed the relationship between landmarks-the fourth to ninth intercostal space (ICS) at the midaxillary line (MAL), through which the distal part of the LTN passes-and the LTN. MATERIALS AND METHODS: We used 25 specimens from 17 embalmed Korean cadavers. The MAL, level of rib and ICS, and regions 5 cm anteroposterior to the MAL (aMAL/pMAL) were established to measure the position of the LTN crossing the MAL, pathway of the LTN, and entering points of the LTN to the SA. RESULTS: The LTN crossed the MAL in 76% of the specimens. The LTN crossed the MAL within the fifth to sixth rib level in 70.8%. Seventy-six percent of the branches entered the SA within the fourth to sixth ICS. The fifth rib and ICS were the most frequent regions aMAL; however, several branches were found pMAL. The LTN entered the SA in 92.6% of the specimens within 3 cm anterior and 1 cm posterior to the MAL. CONCLUSIONS: We set the danger zone as 4 cm near the MAL at the fourth to sixth ICS for thoracotomy. In addition, we proposed the fifth ICS in aMAL at the superficial plane as the alternative injection point for SAPB when blocking the LTN, and the fifth ICS in pMAL at the deep plane to prevent blocking the LTN.

Guideline for Botulinum Toxin Injections in the Effective Treatment of Fibular Spasticity.

This study used a modified Sihler's staining method to analyze the nerve distributions of the fibularis muscle to identify the most effective sites for botulinum toxin injection for fibular spasticity treatment. Ten specimens of the fibularis longus and brevis were obtained bilaterally from five fixed cadavers. The applied method of modified Sihler's staining was designed to reveal the intramuscular nerve distribution of the fibularis muscles. We divided the fibularis muscles into four quarters, which were defined as Sections 1-4 starting from the proximal part of the leg. There were one, two, and three nerve entry points in one (10%), six (60%), and three (30%) of the fibularis longus specimens, respectively, and in four (40%), five (50%), and one (10%) of the fibularis brevis specimens, respectively. We counted the number of nerve endings in each section: 321 and 195 points were identified in the fibularis longus and brevis, respectively. The densities of nerve endings were highest in Section 2 of the fibularis longus (147 of 321, 46%) and in Section 3 of the fibularis brevis (78 of 195, 40%). The landmarks used in this study (the fibular head and lateral malleolus) are easily palpable on the skin's surface, allowing clinicians to target the effective injection site (Section 2) without requiring ultrasound guidance, especially for the fibularis longus. Clin. Anat. 33:365-369, 2020. © 2019 Wiley Periodicals, Inc.

The Nasolabial Fold: A Micro-Computed Tomography Study.

BACKGROUND: The nasolabial fold is known to be a challenging midface feature for aesthetic physicians. However, the steric conformation of the structures related to the nasolabial fold has remained undefined because the composition and topography of this region are highly intricate. Therefore, this study aimed to clarify the three-dimensional structures of the nasolabial fold using micro-computed tomography and verify their detailed composition by means of histologic observation. METHODS: Twenty-four specimens were collected from the area beside the alae nasi to the area above the oral angle of 12 cadavers (mean age, 80.3 years) bilaterally. Twelve specimens were evaluated by means of phosphotungstic acid contrast staining, and the rest were evaluated by means of histologic staining. All specimens were divided into three regions and analyzed comprehensively. RESULTS: The medial region of the nasolabial fold had dense irregular connective tissue intermingled with muscle fibers; the lateral region of the nasolabial fold had numerous fibrous septa with abundant adipose tissue. The levator labii alaeque nasi and the zygomaticus minor were attached to the medial part of the nasolabial fold, and the fascial septa were intermittently tethered to the dermis, lateral to the nasolabial fold. The extension of the adipose tissue within the fascial septa was limited by the lateral border of the muscle attachment. CONCLUSIONS: Dimensional and distributional alterations of the adipose tissues with senescence could render the nasolabial fold deeper by increasing the depth of the subcutaneous layer, lateral to the fold. Thus, to ameliorate the fold, the adipose tissue, lateral to the fold, or the muscle traction, medial to the fold, should be altered.

Influence of injectate volume on paravertebral spread in erector spinae plane block: An endoscopic and anatomical evaluation.

The paravertebral spread that occurs after erector spinae plane block may be volume-dependent. This cadaveric study was undertaken to compare the extent of paravertebral spread with erector spinae plane block using different dye volumes. After randomization, twelve erector spinae plane blocks were performed bilaterally with either 10 ml or 30 ml of dye at the level of T5 in seven unembalmed cadavers except for two cases of unexpected pleural puncture using the 10 ml injection. Direct visualization of the paravertebral space by endoscopy was performed immediately after the injections. The back regions were also dissected, and dye spread and nerve involvement were investigated. A total of five 10 ml injections and seven 30 ml injections were completed for both endoscopic and anatomical evaluations. No paravertebral spread was observed by endoscopy after any of the 10-ml injections. Dye spread to spinal nerves at the intervertebral foramen was identified by endoscopy at adjacent levels of T5 (median: three levels) in all 30 ml injections. In contrast, the cases with two, four, and three out of five were stained at only the T4, T5, and T6 levels, respectively, with the 10 ml injection. Upon anatomical dissection, all blocks were consistently associated with posterior and lateral spread to back muscles and fascial layers, especially with the 30 ml injections, which showed greater dye expansion. In one 30 ml injection, sympathetic nerve involvement and epidural spread were observed at the level of the injection site. Although paravertebral spread following erector spinae plane block increased in a volume-dependent manner, this increase was variable and not pronounced. As the injectate volume increased for the erector spinae blocks, the injectate spread to the back muscles and fascial layers seemed to be predominantly increased compared with, the extent of paravertebral spread.

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue.

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5-7 mm, above which the process was slowed, and the optimum duration was 5-7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Interfascicular septum of the calcaneal tunnel and its relationship with the plantar nerves: A cadaveric study.

The relationship between the plantar nerves and internal fascial structure of the calcaneal tunnel is clinically important to alleviate pain of the sole. The study aimed to investigate the three-dimensional (3D) anatomy of the calcaneal tunnel and its internal fascial septal structure by using microcomputed tomography (mCT) with a phosphotungstic acid preparation, histologic examination, and ultrasound-guided simulation. Twenty-one fixed cadavers and three fresh-frozen cadavers (13 men and 11 women, mean age 82.1 years at death) were used in this study. The 3D images of the calcaneal tunnel harvested by mCT were analyzed in detail. Modified Masson trichrome staining and serial sectional dissection after ultrasound-guided injection were conducted to verify the 3D anatomy. Within the calcaneal tunnel, the interfascicular septum (IFS) commenced proximal to the malleolar-calcaneal line and distal to the bifurcation of the tibial nerve into the plantar nerves. The medial and lateral plantar nerves were separated by the IFS, which divided the calcaneal tunnel into two compartments. The plantar nerves were ramified into two or three branches within each compartment. The IFS terminated around the talocalcaneonavicular joint, and the plantar nerves traveled into the sole. Clinical manipulation of the plantar nerves should be performed in consideration of the fact that they are clearly separated by the IFS. Clin. Anat. 32:877-882, 2019. © 2019 Wiley Periodicals, Inc.

A Detailed Analysis of the Blood Supply to the Subscapularis Muscle.

This study aimed to provide a comprehensive description of the arterial supply to the subscapularis (SSC) muscle. This will provide critical information for various surgical procedures. Ten specimens of embalmed Korean cadavers were dissected and subjected to modified Sihler's method to reveal the branching pattern of the arteries surrounding the subscapularis, and its intramuscular blood supply. The SSC muscle was generally supplied by branches from the subclavian artery (suprascapular artery, supraSA; circumflex scapular artery, CxSA; and dorsal scapular artery, dSA) and the axillary artery (subscapular artery, subSA; lateral thoracic artery, LTA; posterior circumflex humeral artery, PCxHA; and a branch of the axillary artery, AAbr). The anterior aspect of the muscle was supplied by the subSA, LTA, CxSA, supraSA, and AAbr. The posterior aspect of the muscle was supplied by the supraSA, PCxHA, and subSA. The dSA was more scarcely distributed than the other arteries. In two cases, the dSA supplied the portion of the muscle near the medial border of the scapular. The anterior side of the muscle tendon was supplied by the CxSA, and its posterior side was supplied by the PCxHA. The subSA can be considered to be the main branch supplying the SSA based on its distribution area of arteries. It was mostly situated within the lower region of the SSC. After distributing to the anterior surface of the SSC, some branches of the subSA reached the posterior surface as perforating branches. Clin. Anat. 32:642-647, 2019. © 2019 Wiley Periodicals, Inc.

Intramuscular innervation of the subscapularis muscle and its clinical implication for the BoNT injection: An anatomical study using the modified Sihler's staining.

Shoulder pain is commonly associated with spasticity of the rotator cuff muscles including the subscapularis (SSC). The aim of this study was to elucidate the intramuscular innervation pattern of the SSC using the modified Sihler's staining technique to facilitate the targeting of botulinum neurotoxin (BoNT) injections to alleviate shoulder spasticity. Ten SSC specimens (mean age, 81.5 years) were used in this study. Modified Sihler's staining was used to clarify the muscle and to stain the intramuscular nerves. Their extramuscular and intramuscular innervation patterns were examined. The upper subscapular, lower subscapular, thoracodorsal, and axillary nerves (USN, LSN, TDN, and AXN) innervated the SSC in 100%, 80%, 20%, and 40% of specimens, respectively. There was an anastomosis between the USN and LSN in the central portion of the SSC in more than half of the cases. The USN innervated the overall portion of the muscle. In contrast, the additional branches from the TDN and AXN innervated the inferior SSC portion. The superficial branches of the USN were mostly distributed in the superior SSC portion while the deep branches were distributed in the inferior portion. As a major intramuscular nerve within the SSC, the USN should be targeted by a BoNT injection. Regarding the USN distribution, the aim should be to spread the BoNT injectate within the central SSC portion. For supplementary injection to the AXN, the lateral approach would be more appropriate than alternatives. A physician performing a BoNT injection should consider the intramuscular innervation of the SSC portion. Clin. Anat. 32:110-116, 2019. © 2018 Wiley Periodicals, Inc.