Sihler's Staining Technique Research Articles

Intramuscular nerve distribution of the sternocleidomastoid muscle for the botulinum toxin injection.

The aim of this study is to define the intramuscular nerve distribution of the sternocleidomastoid muscle (SCM) and the innervation zones (IZ) to describe the optimal botulinum toxin injection sites. The cricoid cartilage (CC), laryngeal prominence (LP) and hyoid bone (HB) and angle of mandible (AM) were determined as landmarks. The length of the muscles were measured between the sternoclavicular joint and tip of the mastoid process. SCM was evaluated in two parts as anterior and posterior divided by the line where the length of the muscle was measured. Measurements were made to define the relationships of the SCM with common carotid artery, internal and external jugular veins. IZ were described according to these vessels. Afterwards, Modified Sihler's staining technique was applied to expose the intramuscular nerve distribution. The average length of SCM was 160,1mm. Motor entry point of the accessory nerve fibers were between the AM-HB lines, in the range of 30-40% of the muscle length, and in the posterior part of the muscles. IZ were between the HB-CC lines in the anterior and posterior part. When this interval was examined according to the vessels, the optimal injection sites were between the LP-CC lines. This study shows the position of the intramuscular nerve fibers endings of the SCM according to the chosen landmarks and the relationship of the IZ with the vessels to prevent complications. These results can be used as a guide for safe and effective botulinum toxin injections with optimal quantities.

Evaluation of nerve innervation distribution of orbicularis oculi muscle for botulinum neurotoxin application using Sihler's method

BackgroundBlepharospasm is the involuntary and forced closure of the eyelids due to spasm-like contraction of the orbicularis oculi muscle around the eye. The study aimed to reveal the nerve innervation of the orbicularis oculi muscle for Botulinum neurotoxin (BoNT) treatment of blepharospasm by Sihler’s staining technique. MethodsThis study used six cephalus (12 orbicularis oculi muscles) with an average age of 69,6 (62−88) years. Then, the orbicularis oculi muscles were removed by dissection, and Sihler's staining technique was applied to the muscles. The limit for dissection of the muscle is the orbital opening level, where the nerves enter the muscle, and the muscle attaches to the bone. ResultsIn the region above the palpebral fissure, the nerves entering the muscle were densely located in the superolateral quadrant between 9 and 12 o′clock in the right eye and between 12 and 3 o′clock in the left eye. In the region under the palpebral fissure, the nerves’ density in the orbicularis oculi muscle was higher between 4 and 7 o′clock in the right eye and between 5 and 8 o′clock in the left eye. ConclusionThis study unveiled the nerve branches associated with the orbicularis oculi muscle. It pinpointed precise crossing points of these nerve branches, which can be targeted for applying BoNT in treating blepharospasm. Consequently, by utilizing minimal BoNT, it is anticipated that the quantity of toxin administered will decrease, leading to more efficient outcomes and reduced expenses. Furthermore, this approach can aid in averting potential undesired complications during BoNT administration.

Guidance in botulinum neurotoxin injection for lower extremity spasticity: Sihler's staining technique.

Spasticity is a motor disease characterized by a velocity-dependent acceleration in muscle tone or tonic stretch reflexes linked to hypertonia. Lower limb spasticity has been successfully treated with botulinum neurotoxin; however, the injection sites have not been generalized. Sihler's stain has been used to visualize intramuscular nerve distribution to guide botulinum neurotoxin injection. Sihler staining is a whole-mount nerve staining technique that allows visualization of nerve distribution and mapping of entire nerve supply patterns in skeletal muscle with hematoxylin-stained myelinated nerve fibers. This study reviewed and summarized previous lower extremity spasticity studies to determine the ideal injection site for botulinum neurotoxin.

Assessing the innervation of the dorsal wrist capsule using modified Sihler's staining.

The aim of this study was to assess the innervation of the dorsal articular capsule of the wrist using modified Sihler's staining. Thirty dorsal wrist capsules were collected from 15 donors (both sides) within 12 hours of death. All the capsules were collected in the same manner - using the dorsal incision. The specimens were stained according to the protocol of the modified Sihler's staining technique. The preserved capsules were analysed under 8-16× magnification of an optical microscope for the presence of major nerve trunks, their major and minor branches, and nerve connections. The range of innervation visualised was that the posterior interosseous nerve innervated approximately 60% of the central part; the remaining area was innervated by the dorsal sensory branch of the radial nerve and medial antebrachial cutaneous nerve. The constant findings were the branches departing from the ulnar side of the posterior interosseous nerve and from the radial side, with an exception seen in 2 cases. A communicans branch between the posterior interosseous nerve and medial antebrachial cutaneous nerve was seen in all the specimens. The posterior interosseous nerve innervation extended beyond the level of the carpometacarpal joints II-V. The modified Sihler's staining technique allows for transparent visibility of the nerves that innervate the dorsal wrist capsule. However, it does not allow as accurate assessment as does histological examination, especially regarding the evaluation of nerve endings. Nevertheless, this method provides a significantly larger area of nerve observation than is provided by histological examination.

Intramuscular pathway and fascicular characteristics of the segmental intercostal innervation to rectus abdominis.

Rectus abdominis is a muscle that is commonly used clinically as a muscle flap, especially in reconstructive surgery. Its intramuscular innervation, however, has rarely been examined in detail or accurately mapped. The purpose of this study was to use biopsied, histological specimens complemented with a staining technique to investigate intramuscular nerve connections and distribution of the rectus abdominis. Four fresh human cadavers were included in the study and rectus abdominis was dissected bilaterally. Nerve sections innervating the rectus abdominis were biopsied and histologically processed. Sections were viewed under the microscope, and axons within each fascicle were counted using imaging software. All specimens were stained with a modified Sihler's staining technique. Intramuscular innervation was observed and the number as well as distribution was recorded. Macroscopically stained specimens showed that the eighth, ninth, 10th and 12th intercostal nerves innervated the eight muscle bellies of rectus abdominis. The greatest number of minor nerve branching as well as intramuscular nerve communications originated from nerve roots T9 and T10. Minor nerve branches crossed tendinous intersections to communicate with adjacent nerves and innervate adjacent muscle bellies. Nerves originating from T9 had the greatest number of nerve fascicles and the highest axon count in each cadaver. The rectus abdominis is divided into four compartments with each receiving its own independent nerve supply. Minor nerve branches crossed tendinous intersections to communicate with adjacent muscle bellies and nerves suggesting that rectus abdominis can be used as a whole in innervated free flap transfer procedures.

The course of posterior interosseous nerve in the wrist capsule. An anatomical study using the modified Sihler's staining.

Thirty dorsal wrist capsules were collected bilaterally from 15 donors (thirty capsules) within 12 hours of death. By the dorsal incision the capsules were collected in the same manner. The specimens were stained according to the protocol of modified Sihler's staining technique. The preserved capsules were analysed under 8-16× magnification of optical microscope for the presence of major posterior interosseous nerve trunks, their major and minor branches, and nerve connections. Three main types of nerve course were identified within the joint capsule. Type I - the most common, with the presence of a single trunk with the excursion of the first main branch on the radial side, two main branches on the ulnar side, the presence of the prevailing number of small branches on the radial side and the presence of 3-4 branches extending beyond the level of the carpo-metacarpal joints. Type II with the presence of two main nerve trunks, running almost in parallel with the first main branch on the radial side, two main branches on the ulnar side with presence of a predominant number of small branches on the radial side and the presence of 3-4 branches running beyond the level of carpo-metacarpal joints. Type III (least often) with the presence of crossed main nerve trunks. The modified Sihler's staining technique allows for transparent visibility of the nerves innervation the dorsal wrist capsule. However does not allow accurate assessment as histological examination, especially in evaluation of nerve endings, but it gives a significantly larger area of nerve observation.

Intramuscular innervation of the lateral rectus muscle evaluated using sihler's staining technique: Potential application to strabismus surgery.

The latest research suggests that the abducens nerve may be divided into subbranches that reach functionally distinct zones of the lateral rectus muscle. The goal of the study was to examine this muscle's innervation, including the detailed distribution of the intramuscular subbranches of the abducens nerve. Twenty-five lateral rectus muscle specimens were harvested (with the orbital segment of the abducens nerve), fixed in 10% formalin solution, and stained with Sihler's whole mount nerve staining technique. Subbranches running to the lateral rectus divided into two main groups: superior and inferior. Both groups of subbranches are distributed in a fan-shaped manner, show a characteristic "tree-like" branching pattern and form terminal plexus near the proximal half of the lateral rectus muscle. However, some smaller subbranches run as far as the muscle's insertion, and recurrent subbranches also reach its origin. With respect to their course to the muscle's origin or insertion, the smallest subbranches running within the muscle may be associated with innervation of the tendon. In the majority of cases (88%), superior and inferior subbranches of the abducens nerve overlapped in the central one-third of the lateral rectus muscle's width so that any clearly distinct anatomical segments of the muscle could be observed based on Sihler's technique. In the remaining 12% of specimens, superior and inferior groups of subbranches innervated two distinct compartments of the lateral rectus muscle with no overlapping. Dense, fan-shaped distribution of abducens nerve intramuscular subbranches can be observed within the lateral rectus muscle. Clin. Anat. 33:585-591, 2020. © 2019 Wiley Periodicals, Inc.

Hip Adductor Intramuscular Nerve Distribution Pattern of Children: A Guide for BTX-A Treatment to Muscle Spasticity in Cerebral Palsy.

To investigate the intramuscular nerve distribution pattern in the hip adductors of children and to precisely locate the injection site for botulinum toxin type A (BTX-A) as a treatment for hip adductor spasticity in children with cerebral palsy. Modified Sihler's whole mount nerve staining technique was employed to observe the distribution of intramuscular nerves in hip adductors of children and to further locate zones where terminal nerves are concentrated. The terminal nerves of the adductor longus appeared in a longitudinal distribution band parallel to the line between the upper 1/3 point of the lateral boundary and the center of the medial boundary. In adductor brevis, the terminal nerves showed a sheet-like distribution with a nerve dense area located in the middle of the muscle belly that extends from the upper-inner region to the lower-outer region. Gracilis showed a dense area of terminal nerves in the middle of the muscle belly, closer to the posterior boundary. In adductor magnus, the dense area of terminal nerves showed a sheet-like distribution in the middle and lower region of the muscle belly. The dense area of terminal nerves in the pectineus was located in the middle of the muscle belly. This study is the first to systematically investigate the intramuscular nerve distribution pattern in the hip adductors. The results indicated that the best targets for BTX-A injection, when treating spasticity, are the dense regions of terminal nerves described above.

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.

Method to test the long-term stability of functional electrical stimulation via multichannel electrodes (e.g., applicable for laryngeal pacing) and to define best points for stimulation: in vivo animal analysis.

The study aim was to identify and analyze intramuscular electrically sensitive points. Electrically sensitive points are herein defined as positions, which allow muscles stimulation with a minimum possible fatigue for a maximum amount of time. A multichannel array electrode was used which could be interesting to retain the function of larynx muscle after paralysis. Eight array electrodes were implanted in the triceps brachii muscle of four rats. While being under anesthesia, the animals were intramuscularly stimulated at 16 different positions. Sihler's staining technique was used to make visible the nerves routes and the intramuscular position of the individual electrode plate. The positions of the motor end plates were determined by means of multichannel-electromyography. The positions that allow longest stimulation periods are located close to the points where the nerves enter the muscle. Stimulation at the position of the motor end plates does not result in stimulation periods above average. Locations initially causing strong muscle contractions are not necessarily identical to the ones allowing long stimulation periods. The animal model identified the stimulation points for minimal possible muscle fatigue stimulation as being located close to the points of entrance of the nerve into the muscle. Stimulation causing an initially strong contraction response is no indication of optimal location of the stimulation electrode in terms of chronic stimulation. The array electrode of this study could be interesting as a stimulation electrode for a larynx pacemaker.

Intramuscular innervations of lower leg skeletal muscles: applications in their clinical use in functional muscular transfer.

This study aims to investigate nerve distribution patterns of human lower leg skeletal muscles using a modified Sihler's staining method. Sixteen lower leg from eight fresh adult cadavers were used in this study and all the skeletal muscles were dissected. The muscle specimens were classified according to Lim's classification. The specimens were then stained by further modified Sihler's staining technique. Data were analyzed according to research results. After the staining, we found four patterns of nerve distribution in human lower leg muscles: (1) Type 1: single nerve pattern in which the nerve branches into two either running parallel to each other or radiating in a spray pattern (such as the extensor digitorum longus, extensor hallucis longus, fibularis brevis and flexor hallucis longus). (2) Type 2: double nerve pattern, one being proximal and the other being distal (such as the extensor digitorum longus, flexor digitorum longus, flexor hallucis longus). (3) Type 3: multiple branch pattern (such as the tibialis anterior, fibularis longus, gastrocnemius, soleus, tibialis anterior and popliteus). Our modified Sihler's staining method is useful for research of large muscles and intramuscular nerves in human. These findings might provide guidance for clinicians for muscle reconstruction surgery.

Three-dimensional observation of the mandible and accessory mental nerve using Sihler's staining technique

It is generally known that there are two mental foramina and two mandibular foramina. It is also known that occasionally there are some accessory foramina in the mandible, including lingual, buccal, retromolar, and accessory mental foramina. Among these accessory foramina, the accessory mental foramen in particular is located in a dangerous area for the oral surgeon1).The reported incidence of an accessory mental foramen ranges from 2.0% to 11.9%. We can easily diagnose accessory mental foramina using three-dimensional computed tomography (3D-CT). However, it is sometimes difficult to diagnose the accessory mental foramina using panoramic findings alone2). There are few reports of accessory mental nerves branching off the accessory mental foramina.In this study, we investigated the distribution patterns of the accessory mental nerve using Sihler's staining and transparency technique and 3D-CT images. We were able to observe the relations among the mandible and the accessory mental foramen and the accessory mental nerve

Architectural properties of the neuromuscular compartments in selected forearm skeletal muscles.

The purposes f this study were to (i) explore the possibility of splitting the selected forearm muscles into separate compartments in human subjects; (ii) quantify the architectural properties of each neuromuscular compartment; and (iii) discuss the implication of these properties in split tendon transfer procedures. Twenty upper limbs from 10 fresh human cadavers were used in this study. Ten limbs of five cadavers were used for intramuscular nerve study by modified Sihler's staining technique, which confirmed the neuromuscular compartments. The other 10 limbs were included for architectural analysis of neuromuscular compartments. The architectural features of the compartments including muscle weight, muscle length, fiber length, pennation angle, and sarcomere length were determined. Physiological cross-sectional area and fiber length/muscle length ratio were calculated. Five of the selected forearm muscles were ideal candidates for splitting, including flexor carpi ulnaris, flexor carpi radials, extensor carpi radialis brevis, extensor carpi ulnaris and pronator teres. The humeral head of pronator teres contained the longest fiber length (6.23 ± 0.31 cm), and the radial compartment of extensor carpi ulnaris contained the shortest (2.90 ± 0.28 cm). The ulnar compartment of flexor carpi ulnaris had the largest physiological cross-sectional area (5.17 ± 0.59 cm(2)), and the ulnar head of pronator teres had the smallest (0.67 ± 0.06 cm(2)). Fiber length/muscle length ratios of the neuromuscular compartments were relatively low (average 0.27 ± 0.09, range 0.18-0.39) except for the ulnar head of pronator teres, which had the highest one (0.72 ± 0.05). Using modified Sihler's technique, this research demonstrated that each compartment of these selected forearm muscles has its own neurovascular supply after being split along its central tendon. Data of the architectural properties of each neuromuscular compartment provide insight into the 'design' of their functional capability. In addition to improving our understanding of muscle anatomy and function, elucidation of forearm neuromuscular compartments architecture may ultimately provide information useful for selection of muscle subdivisions used in tendon transfer.

Topography of the greater palatine artery and the palatal vault for various types of periodontal plastic surgery

The purpose of the present study is to provide useful data that could be applied to various types of periodontal plastic surgery by detailing the topography of the greater palatine artery (GPA), looking in particular at its depth from the palatal masticatory mucosa (PMM) and conducting a morphometric analysis of the palatal vault. Forty-three hemisectioned hard palates from embalmed Korean adult cadavers were used in this study. The morphometry of the palatal vault was analyzed, and then the specimens were decalcified and sectioned. Six parameters were measured using an image-analysis system after performing a standard calibration. In one specimen, the PMM was separated from the hard palate and subjected to a partial Sihler's staining technique, allowing the branching pattern of the GPA to be observed in a new method. The distances between the GPA and the gingival margin, and between the GPA and the cementoenamel junction were greatest at the maxillary second premolar. The shortest vertical distance between the GPA and the PMM decreased gradually as it proceeded anteriorly. The GPA was located deeper in the high-vault group than in the low-vault group. The premolar region should be recommended as the optimal donor site for tissue grafting, and in particular the second premolar region. The maximum size and thickness of tissue that can be harvested from the region were 9.3 mm and 4.0 mm, respectively.

Anatomy of the infratemporal crest: Implications for cross-facial nerve grafting in temporal myoplasty

Temporalis transfers for reanimation in facial palsy have been criticised for the lack of neural input from a functioning facial nerve. Cross-facial nerve grafting to the deep temporal nerves may provide a solution. An anatomical study was performed to further elucidate the deep temporal nerves (DTN) and arteries, examining anatomical variation and infratemporal distribution. Seventeen temporalis muscles were dissected from 13 cadavers as part of a BSc project. The number, branching pattern, length and diameter of DTN were recorded. Arteries supplying the deep surface of temporalis and their relation to DTN were noted. Six specimens were processed using Sihler's staining technique. Arteries were injected with Iodixanol X-ray contrast medium and radiographs taken. All specimens displayed a single DTN originating from the anterior branch of V(3). A mean of 3 branches was observed. The nerve length was 14.22±3.95 mm. The point of entry of DTN into temporalis showed great consistency. Upon exiting the infratemporal fossa, the posterior deep temporal artery was deep and posterior to DTN in 65% of specimens. The branching pattern of DTN can be classified into three types. The deep arterial supply to temporalis was constant in all specimens. This study provides an anatomical basis for the planning and execution of cross-facial nerve grafting to temporalis, and for protection of vital structures. Furthermore, it helps to clarify inconsistencies in the literature regarding nomenclature of the nerve branching pattern of the deep temporal nerves.

Clinical and anatomical approach using Sihler's staining technique (whole mount nerve stain)

Sihler's staining allows visualization of the nerve distribution within soft tissues without extensive dissection and does not require slide preparation, unlike traditional approaches. This technique can be applied to the mucosa, muscle, and organs that contain myelinated nerve fibers. In particular, Sihler's technique may be considered the best tool for observing nerve distribution within skeletal muscles. The intramuscular distribution pattern of nerves is difficult to observe through manual manipulation due to the gradual tapering of nerves toward the terminal end of muscles, so it should be accompanied by histological studies to establish the finer branches therein. This method provides useful information not only for anatomists but also for physiologists and clinicians. Advanced knowledge of the nerve distribution patterns will be useful for developing guidelines for clinicians who perform operations such as muscle resection, tendon transplantation, and botulinum toxin injection. Furthermore, it is a useful technique to develop neurosurgical techniques and perform electrophysiological experiments. In this review, Sihler's staining technique is described in detail, covering its history, staining protocol, advantages, disadvantages, and possible applications. The application of this technique for determining the arterial distribution pattern is also described additionally in this study.

Intramuscular innervations of muscle flaps that are commonly used in clinical settings

Abductor hallucis, latissimus dorsi, gracilis, rectus abdominis, sartorius and pectoralis minor are muscle flaps that are commonly used in clinic, but their intramuscular innervation has seldom been systematically investigated. Five Chinese fresh human cadavers were included in the study and abductor hallucis, latissimus dorsi, gracilis, rectus abdominis, sartorius and pectoralis muscles were dissected. After gross anatomy measurement, the specimens were then stained by Sihler's staining technique. Intramuscular innervation was observed and the number as well as distribution was recorded. Intramuscular nerves were clearly visualized by Sihler's staining technique. Based on the shape and muscle-tendon morphology, Lim et al. in Muscle Nerve 29:523-530, 2004 grouped the muscles into trapezoidal-shaped (type I), spindle-shaped (type II), and combination-shaped (type III). According to Lim's study the abductor hallucis was a type IIb muscle and was divided into two compartments by the distal tendon. Latissimus dorsi was a type I muscle, divided into 3-4 compartments by intramuscular nerve branches. Gracilis was a type IIa muscle and the distal part was divided into two compartments by intramuscular nerve branches. Rectus abdominis was a type III muscle and the four bellies comprised four compartments, each of which could be designated as a subunit. Sartorius was a type II muscle and it could be divided into 2-3 compartments along the long axis. Pectoralis minor was a type I muscle that was divided into two compartments by extramuscular terminal nerves. The six muscles are divided into several compartments by the tendon or nerve branches, and all of them make good donor tissue for muscle compartment transfer in reconstructive surgery.

Sihler's whole mount nerve staining technique: a review

Sihler's stain is a whole mount nerve staining technique that renders other soft tissue translucent or transparent while staining the nerves. It permits mapping of entire nerve supply patterns of organs, skeletal muscles, mucosa, skin, and other structures after the specimens are fixed in neutralized formalin, macerated in potassium hydroxide, decalcified in acetic acid, stained in Ehrlich's hematoxylin, destained in acetic acid, and cleared in glycerin. The unique advantage of Sihler's stain over other anatomical methods is that all the nerves within the stained specimen can be visualized in their three-dimensional positions. To date, Sihler's stain is the best tool for demonstrating the precise intramuscular branching and distribution patterns of skeletal muscles, which are important not only for anatomists, but also for physiologists and clinicians. Advanced knowledge of the neural structures within mammalian skeletal muscles is critical for understanding muscle functions, performing electrophysiological experiments and developing novel neurosurgical techniques. In this review, Sihler's stain is described in detail and its use in nerve mapping is surveyed. Special emphasis is placed on staining procedures and troubleshooting, strengths and limitations, applications, major contributions to neuroscience, physiological and clinical significance, and areas for further technical improvement that deserve future research.

Sihler's whole mount nerve staining technique: a review

Sihler's whole mount nerve staining technique: a review

The internal innervation and morphology of the human female levator ani muscle

The purpose of this study was to describe the microinnervation of the human the female levator ani muscle complex. Detailed microdissection of 10 fresh female levator ani muscles was performed. Specimens were processed by modified Sihler's staining technique. A composite drawing of the levator ani nerve (LAN) distribution was created. Six hemi-levator ani muscles were stained and digitally reconstructed. The LAN traveled perpendicular to major muscular bundles while progressively branching into finer nerves that eventually entered single muscle fascicles. The LAN continued its course through the iliococcygeous muscle to innervate the puborectalis, puboperinealis, and puboanalis muscles. There was no distinct separation between pubovisceralis and iliococcygeous fibers. There were muscle fibers that could be best described as coccygeoperinealis. The utilized staining technique enabled us to visualize the LAN microinnervation of the levator ani muscle that extends to puboperinealis and puboanalis muscles. A blueprint for LAN and levator ani morphology was created.