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Evaluating intramuscular neural distribution in the cricopharyngeus muscle for injecting botulinum toxin

  • Bo Hae Kim
    Correspondence
    Corresponding author at: Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk University Ilsan Hospital, Dongguk-ro 27, Ilsandong-gu, Goyang 10326, South Korea
    Affiliations
    Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk university Ilsan Hospital, College of Medicine, Goyang, South Korea

    Sensory Organ Research Institute, College of Medicine, Dongguk University, Gyengju, South Korea
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  • Do Hyung Kim
    Affiliations
    Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk university Ilsan Hospital, College of Medicine, Goyang, South Korea
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  • Ji-Hyun Lee
    Affiliations
    Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identification Research Center, College of Dentistry, Yonsei University, Seoul, South Korea
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  • Hyung-Jin Lee
    Affiliations
    Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identification Research Center, College of Dentistry, Yonsei University, Seoul, South Korea
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  • Hee-Jin Kim
    Correspondence
    Corresponding author at: Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identifcation Research Institute, BK21 PLUS Project, Yonsei University College of Dentistry, Room 6 01, 50 Yonsei‑ro, Seodaemun‑gu, Seoul 03722, South Korea
    Affiliations
    Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identification Research Center, College of Dentistry, Yonsei University, Seoul, South Korea
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      Abstract

      Objectives

      The objective of this study was to determine the area in the cricopharyngeus muscle (CPM) where botulinum neurotoxin (BoNT) can be injected safely and effectively by evaluating neural distribution in the CPM.

      Methods

      Eleven specimens of the CPM were gathered from human cadavers. The anatomical relationship between the posterior cricoarytenoid muscle (PCAM) and the CPM was evaluated. Myelinated nerve fibers in the CPM were stained using modified Sihler's method. The CPM was classified into five zones according to the area accessible within the CPM via transcutaneous and transluminal approaches for BoNT injection as follows: i) lateral area including upper area (zone 1) and lower area (zone 2); ii) posterolateral area including upper area (zone 3) and lower area (zone 4); and iii) posterior area (zone 5). Neural distribution originating from the pharyngeal plexus and the extralaryngeal branches of recurrent laryngeal nerve (EBRLN) within each classified zone in stained specimens was determined.

      Results

      Six specimens (12 lateral areas, 12 posterolateral areas, and 6 posterior areas) were suitable for evaluating neural distribution within the CPM. Zone 1 was adjacent to the PCAM the most in all specimens. Nerve endings originating from the EBRLN were observed on four sides of zone 2 (33.3%, 4/12 sides) in three specimens (3/6, 50%). Neural distribution originating from the pharyngeal plexus was found on ten sides (83.3%, 10/12 sides) of zone 3 in five specimens (83.3%, 5/6 specimens) and on nine sides (75.0%, 9/12 sides) of zone 4 in five specimens (83.3%, 5/6 specimens).

      Conclusion

      The posterolateral area (zone 3 and zone 4) is thought to be the most suitable area for alleviating the spasticity of CPM with a minimum dose of BoNT.

      Keywords

      1. Introduction

      The cricopharyngeus muscle (CPM) is a semi-circular muscle attached to the cricoid cartilage. It has a horizontal nerve fiber with a height of 1.5 - 1.9 cm [
      • Sivarao DV
      • Goyal RK.
      Functional anatomy and physiology of the upper esophageal sphincter.
      ,
      • Kelly JH
      • Kuncl RW.
      Myology of the pharyngoesophageal segment: gross anatomic and histologic characteristics.
      . The CPM mainly contributes to pressure generation of the upper esophageal sphincter (UES) by circumferential contraction. Relaxation of the CPM is chiefly regulated by the pharyngoesophageal nerve (PEN) originated from the vagus nerve [
      • Sivarao DV
      • Goyal RK.
      Functional anatomy and physiology of the upper esophageal sphincter.
      ]. Impaired relaxation of the CPM can lead to an insufficient transition of a bolus from the pharynx to the esophagus, resulting in cricopharyngeal dysphagia (CPD) [
      • Kocdor P
      • Siegel ER
      • Tulunay-Ugur OE.
      Cricopharyngeal dysfunction: A systematic review comparing outcomes of dilatation, botulinum toxin injection, and myotomy.
      ]. Since botulinum neurotoxin (BoNT) can reduce the spasticity of the target muscle by decreasing acetylcholine release at the neuromuscular junction [
      • Pirazzini M
      • Rossetto O
      • Eleopra R
      • Botulinum Montecucco C.
      Neurotoxins: Biology, Pharmacology, and Toxicology.
      ], BoNT has been used for alleviating spasticity of the CPM in patients with CPD [
      • Kelly EA
      • Koszewski IJ
      • Jaradeh SS
      • Merati AL
      • Blumin JH
      • Bock JM
      Botulinum toxin injection for the treatment of upper esophageal sphincter dysfunction.
      ].
      Previous studies were focused on the clear identification of the location and boundaries of the CPM for accurately inserting the needle into the muscle itself because this muscle has a small volume, and this effort was made to establish techniques of injecting BoNT into the CPM [
      • Parameswaran MS
      • Soliman AM
      Endoscopic botulinum toxin injection for cricopharyngeal dysphagia.
      ,
      • Zaninotto G
      • Marchese-Ragona R
      • Briani C
      • Costantini M
      • Rizzetto C
      • Portale G
      • et al.
      The role of botulinum toxin injection and upper esophageal sphincter myotomy in treating oropharyngeal dysphagia.
      ,
      • Kim MS
      • Kim GW
      • Rho YS
      • Kwon KH
      • Chung EJ.
      Office-based Electromyography-guided Botulinum Toxin Injection to the Cricopharyngeus Muscle: Optimal Patient Selection and Technique.
      ,
      • Chen JM
      • Chen YJ
      • Ni J
      • Wang ZY.
      Ultrasound, electromyography, and balloon guidance for injecting botulinum toxin for cricopharyngeal achalasia: A case report.
      ]. The posterior bend of UES formed by contracted CPM is used as an anatomical landmark during a transluminal approach for injecting BoNT [
      • Parameswaran MS
      • Soliman AM
      Endoscopic botulinum toxin injection for cricopharyngeal dysphagia.
      ]. Ultrasonography and electromyography can assist localization of the CPM during a transcutaneous approach [
      • Zaninotto G
      • Marchese-Ragona R
      • Briani C
      • Costantini M
      • Rizzetto C
      • Portale G
      • et al.
      The role of botulinum toxin injection and upper esophageal sphincter myotomy in treating oropharyngeal dysphagia.
      ,
      • Kim MS
      • Kim GW
      • Rho YS
      • Kwon KH
      • Chung EJ.
      Office-based Electromyography-guided Botulinum Toxin Injection to the Cricopharyngeus Muscle: Optimal Patient Selection and Technique.
      ,
      • Chen JM
      • Chen YJ
      • Ni J
      • Wang ZY.
      Ultrasound, electromyography, and balloon guidance for injecting botulinum toxin for cricopharyngeal achalasia: A case report.
      ]. However, localization of the CPM using various techniques as mentioned above cannot completely prevent complications following BoNT injection, especially impaired movement of vocal folds [
      • Kelly EA
      • Koszewski IJ
      • Jaradeh SS
      • Merati AL
      • Blumin JH
      • Bock JM
      Botulinum toxin injection for the treatment of upper esophageal sphincter dysfunction.
      ]. Unpredictable diffusion and axonal transport of BoNT are known as key mechanisms of complications following a BoNT injection [
      • Ramirez-Castaneda J
      • Jankovic J
      • Comella C
      • Dashtipour K
      • Fernadez HH
      • Mari Z.
      Diffusion, spread, and migration of botulinum toxin.
      ]. Diffusion and axonal transport of BoNT can be minimized by using a small dose of BoNT. BoNT should be injected into an area where neuromuscular junctions are abundant in order to have sufficient muscle relaxation with a minimum dose of toxin [
      • Ramirez-Castaneda J
      • Jankovic J
      • Comella C
      • Dashtipour K
      • Fernadez HH
      • Mari Z.
      Diffusion, spread, and migration of botulinum toxin.
      ,
      • Yi KH
      • Lee HJ
      • Choi YJ
      • Lee JH
      • Hu KS
      • Kim HJ.
      Intramuscular Neural Distribution of Rhomboid Muscles: Evaluation for Botulinum Toxin Injection Using Modified Sihler's Method.
      .
      The objective of this study was to determine the area where BoNT could be injected safely and effectively by evaluating neural distribution in CPM using a modified Sihler's method to stain myelinated nerve fibers.

      2. Materials and Methods

      Dissection of cadavers was conducted according to the Declaration of Helsinki after obtaining consent and approval from families of cadavers. We obtained approval for anatomical research from our institution (approval number: 22-005). A total of 11 specimens were obtained from Korean cadavers (seven men and four women). The larynx was harvested, including CPM, constrictor muscles, and recurrent laryngeal nerve. The anatomical relationship of CPM with the posterior cricoarytenoid muscle (PCAM) was determined in the process of separating the CPM from the laryngeal framed work. Extralaryngeal branches of the recurrent laryngeal nerve (EBRLN) and the constrictor muscles were attached to the side of the CPM. These harvested structures were simultaneously stained by the modified Sihler method. Neural distribution originated from the pharyngeal plexus and the EBRLN within each classified zone in stained specimens was determined. The presence of neural distribution within each zone was determined when neural arborization pattern or nerve ending was observed in classified zone.

      2.1 Classification of cricopharyngeus muscle

      CPM was classified into five zones in detail considering accessibility of the area by transluminal and transcutaneous approaches. Anatomical relationship of the CPM with major structures related to vocal fold immobility following a BoNT injection was also evaluated (Table 1). Lateral and posterolateral areas of the CPM were accessible areas via the conventional transcutaneous approach (Fig. 1A and B). A transluminal approach could access posterolateral and posterior area of the CPM (Fig. 1C and D). The most prominently palpated upper and lower points of the ventral surface of the cricoid cartilage are anatomical landmarks during transcutaneous injection [
      • Kim MS
      • Kim GW
      • Rho YS
      • Kwon KH
      • Chung EJ.
      Office-based Electromyography-guided Botulinum Toxin Injection to the Cricopharyngeus Muscle: Optimal Patient Selection and Technique.
      ]. Therefore, neural distribution in the CPM was limitedly evaluated between imaginary horizontal lines parallel to the most definitely palpated upper and lower point at the ventral surface of the cricoid cartilage (Fig. 2A and B). The boundary between lateral and posterolateral areas was the dorsal end of the cricoid cartilage. This boundary was the point where the rotating of the larynx and the bending of the needle were required to advance the needle from the lateral area to the posterolateral area during a transcutaneous approach (Fig. 1A) [
      • Kim MS
      • Kim GW
      • Rho YS
      • Kwon KH
      • Chung EJ.
      Office-based Electromyography-guided Botulinum Toxin Injection to the Cricopharyngeus Muscle: Optimal Patient Selection and Technique.
      ]. Lateral and posterolateral areas were divided into two zones using a horizontal line at the mid-height parallel to upper and lower boundaries of the cricoid cartilage. The lateral area was classified into zone 1 (upper zone of the lateral area) and zone 2 (lower zone of the lateral area). The posterolateral area was divided into the upper zone as zone 3 and the lower zone as zone 4 (Fig. 3). The posterior area (zone 5) was defined as a space of 1 cm between both posterolateral areas where BoNT injection was only accessible via the transluminal approach (Fig. 3). Posterolateral and posterior areas formed the posterior bend of the CPM during a transluminal approach (Fig 1B) [
      • Chiu MJ
      • Chang YC
      • Hsiao TY.
      Prolonged effect of botulinum toxin injection in the treatment of cricopharyngeal dysphagia: case report and literature review.
      ].
      Table 1Anatomical location between each zone and main structures associated with vocal fold movement.
      ZoneAnatomical relationshipAvailable approach
      EBRLNPCAM
      1Relatively closeCloseTranscutaneous (before reaching the vertebra)
      2PresenceRelatively closeTranscutaneous (before reaching the vertebra)
      3DistantRelatively distant1. Transluminal

      2. Transcutaneous (after reaching the vertebra)
      4Relatively distantDistant1. Transluminal

      2. Transcutaneous (after reaching the vertebra)
      5DistantDistantTransluminal
      Fig. 1
      Fig. 1Brief presentation of techniques of injecting botulinum toxin (BoNT) into the cricopharyngeus muscle (CPM) reported in previous studies. (A) Lateral and posterolateral areas of the CPM are accessible areas (yellow circle) via a transcutaneous approach for BoNT injection. (B) Simulation of transcuatenous approach on specimen before staining. (C) Transluminal approach is accessible into posterolateral and posterior areas (yellow circle) of the CPM. (D) The posterior bend formed by contracted CPM is an anatomical landmark for injecting BoNT into the CPM (yellow circle) during a transluminal approach.
      Abbreviations: CPM, cricopharyngeus muscle; PCAM, posterior cricoarytenoid muscle.
      Fig. 2
      Fig. 2Boundaries for evaluating neural distribution within the cricopharyngeus muscle (CPM). (A) Silk tagging sutures (white circles) for marking the boundary between lateral and posterolateral areas (white dot line) of the CPM before detaching cartilaginous structures from the CPM. (B) Boundaries of evaluation of neural distribution within the CPM on the stained specimen. Red dotted lines were boundaries between lateral and posterolateral areas determined by silk tagging sutures. Although the black line was actual upper and lower boundaries of the CPM, evaluation of neural distribution within the CPM was limited between prominently palpated upper and lower points (dotted lines) of the ventral surface of the cricoid cartilage considering the technique of a transcutaneous approach.
      Abbreviations: ICM, inferior constrictor muscle.
      Fig. 3
      Fig. 3Classification of the cricopharyngeus muscle (CPM) into five zones in detail considering the area accessible by transluminal and transcutaneous approaches. (A) The lateral area was classified into two zones, the upper area as zone 1 (white box) and the lower area as zone 2 (yellow box). The mid-height between prominently palpated upper and lower points of the ventral surface of cricoid cartilage was the boundary between the two zones. (B) The posterolateral area was also subclassified into two zones by an imaginary line extending from the lateral area (upper area, zone 3 (orange box); lower area, zone 4 (blue box)). The posterior area (purple box) of the CPM was defined as a space of about 1 cm between posterolateral areas on both sides, without subclassification. (C) Axial image of the CPM.

      2.2 Cadaver dissection

      Since cartilaginous structures must be removed in order to stain the intramuscular nerve with Sihler's method, the boundary between lateral and posterolateral areas was marked using two silk sutures before detaching muscles from the laryngeal framework. The first marking was made at the junction between the upper boundary and the dorsal end of the cricoid cartilage. The second marking was made at the constrictor muscle overlying the dorsal margin of the thyroid cartilage. The imaginary line between two markings was the boundary between lateral and posterolateral areas of the CPM (Fig. 2A). The EBRLN innervating the CPM was separated from the laryngeal framework by cutting down the intralaryngeal branch of recurrent laryngeal nerve at the laryngeal inlet of recurrent laryngeal nerve. After detaching the CPM and constrictor muscles from cartilaginous structures, the anatomical relationship between the CPM and the PCAM was evaluated. We divided the esophagus at the ventral portion and then removed the inner mucosa of the esophagus.

      2.3 Modified Sihler's staining

      Modified Sihler staining of the CPM known to stain myelinated nerve fibers was performed to identify neural distribution as suggested in previous study [
      • Yi KH
      • Lee HJ
      • Choi YJ
      • Lee JH
      • Hu KS
      • Kim HJ.
      Intramuscular Neural Distribution of Rhomboid Muscles: Evaluation for Botulinum Toxin Injection Using Modified Sihler's Method.
      ]. The intramuscular nerve was observed as a blue line. The first step of the modified Sihler's method was fixing the CPM with 10% un-neutralized formaldehyde for a month. Formaldehyde was sometimes changed to maintain transparency. The fixed CPM was macerated with running water for an hour. Depigmentation of the CPM was then performed using 3% aqueous potassium hydroxide in combination with hydrogen peroxide for three weeks. The next process of CPM was decalcification of the specimen using Sihler I solution (a mixture of glacial acetic acid, aqueous chloral hydrate, and glycerin) for three days. For up to four weeks, the decalcified specimen was stained with Sihler II solution (a mixture of aqueous chloral hydrate, glycerin, and acetic acid). After finishing staining the CPM, the muscle fiber of the CPM was destained using Sihler I solution for an hour and then neutralized in running tap water for 30 minutes. The CPM was finally immersed in 0.05% lithium carbonate to stain nerve fibers. It was then cleaned with formamide for 100 hours [
      • Yi KH
      • Lee HJ
      • Choi YJ
      • Lee JH
      • Hu KS
      • Kim HJ.
      Intramuscular Neural Distribution of Rhomboid Muscles: Evaluation for Botulinum Toxin Injection Using Modified Sihler's Method.
      ].

      3. Results

      Among 11 specimen of CMP, between zones located in the lateral area of the CPM, zone 1 was adjacent to the fascia of the PCAM the most in all specimens (Fig. 4A). Five specimens were unsuitable for the evaluation of intramuscular neural distribution because the nerve fiber could not be clearly distinguished from the muscle fiber.123
      Fig. 4
      Fig. 4Anatomical relationship of the cricopharyngeus muscle (CPM) with major structures that can cause vocal fold immobility following botulinum toxin injection. (A) The upper zone of the lateral area (Zone 1) of the CPM is located adjacent to the posterior cricoarytenoid muscle (PCAM) the most (the red circle indicates the junction area between the CPM and PCAM). (B). Neural distribution within the cricopharyngeus muscle (zone 2) originated from the extralaryngeal branch of recurrent laryngeal nerve (white arrow) on converted image.
      Abbreviations: CPM, cricopharyngeus muscle; PCAM, posterior cricoarytenoid muscle.
      Therefore, the evaluation of neural distribution in the CPM was available in six specimens (12 lateral areas, 12 posterolateral areas, and 6 posterior areas). Nerve endings originated from the EBRLN were observed on four sides (33.3%, 4/12 sides) of three specimens (3/6, 50%) (Fig. 4B). Neural distribution from the EBRLN was not found in other zones (Table 2).
      Table 2Neural distribution originated from the pharyngeal plexus and extralaryngeal branch of the recurrent laryngeal nerve in each zone.
      SpecimenSuitabilityPresence of neural distribution
      No.RightLeft
      Pharyngeal plexusEBRLNPharyngeal plexusEBRLN
      1YesZone 1 and 2NoneZone 1Zone 2
      2YesZone 1-4NoneZone 1-4Zone 2
      3YesZone 3-4NoneZone 3-4None
      4YesZone 3-4NoneZone 3-4None
      5YesZone 3-4Zone 2Zone 3-4Zone 2
      6NoN/AN/AN/AN/A
      7NoN/AN/AN/AN/A
      8NoN/AN/AN/AN/A
      9NoN/AN/AN/AN/A
      10NoN/AN/AN/AN/A
      11YesZone 3-4NoneZone 3None
      Neural distribution originated from the pharyngeal plexus was found on four sides (33.3%, 4/12 sides) of two specimens (33.3%, 2/6 specimens) in zone 1 and on three sides (25.0%, 3/12 sides) of three specimens (50%, 3/6 specimens) in zone 2. The posterolateral area was the most abundant area of neural distribution that originated from the pharyngeal plexus. Neural distribution was found on ten sides (83.3%, 10/12 sides) of five specimens (83.3%, 5/6 specimens) in zone 3 and on nine sides (75.0%, 9/12 sides) of five specimens (83.3%, 5/6 specimens) (Fig. 5A and B). Although the posterior area (zone 5) was far from both the PCAM and EBRLN the most among five zones, there was no gross neural distribution originated from the pharyngeal plexus or EBRLN in zone 5 (0%, 0/6). These results of neural distribution in each zone obtained from six specimens are summarized in Tables 2 and 3.
      Fig. 5
      Fig. 5Neural distribution within the cricopharyngeus muscle (CPM) originated from the pharyngeal plexus. (A). The presence of neural arborization or gross nerve ending originated from the pharyngeal plexus within zone 1-2 on the right side and zone 1 on the left side. The ending of extralaryngeal branch of recurrent laryngeal nerve (EBRLN) was found on zone 2 of left side. (B). Presence of neural arborization or gross nerve ending within zones 3 and 4 on both sides.
      Table 3Proportion of the presence of neural distribution in specimens (neural arborization or ending originated from the pharyngeal plexus was the most frequently observed in Zone 3 (10/12, 83.3%), which was the upper zone of the posterolateral area closed to the posterior cricoarytenoid muscle (PCAM) but relatively far from the ending of the extralaryngeal branch of RLN (EBRLN)).
      Zone 1Zone 2Zone 3Zone 4Zone 5
      Pharyngeal plexus
      Specimen2 / 6 (33.3 %)3 / 6 (50.0 %)5 / 6 (83.3 %)5 / 6 (83.3 %)0 / 6 (0 %)
      Side4 / 12 (33. 3 %)3 / 12 (25. 0 %)10 / 12 (83. 3 %)9 / 12 (75. 0 %)0 / 6 (0 %)
      EBRLN
      Specimen0/6 (0 %)3 / 12 (25. 0 %)0 / 6 (0 %)0 / 6 (0 %)0 / 6 (0 %)
      Side0 / 12 (0 %)4 / 12 (33. 3 %)0 / 12 (0 %)0 / 12 (0 %)0 / 6 (0 %)

      4. Discussion

      Although BoNT has been commonly used for treating CPD, the injection point and dose of BoNT into the CPM vary according to physicians [
      • Kelly EA
      • Koszewski IJ
      • Jaradeh SS
      • Merati AL
      • Blumin JH
      • Bock JM
      Botulinum toxin injection for the treatment of upper esophageal sphincter dysfunction.
      ,
      • Kim DY
      • Park CI
      • Ohn SH
      • Monn JY
      • Chang WH
      • Park SW.
      Botulinum toxin type A for poststroke cricopharyngeal muscle dysfunction.
      . Reported complication rate also varies even if the same method has been used [
      • Kelly EA
      • Koszewski IJ
      • Jaradeh SS
      • Merati AL
      • Blumin JH
      • Bock JM
      Botulinum toxin injection for the treatment of upper esophageal sphincter dysfunction.
      ,
      • Kim DY
      • Park CI
      • Ohn SH
      • Monn JY
      • Chang WH
      • Park SW.
      Botulinum toxin type A for poststroke cricopharyngeal muscle dysfunction.
      . Therefore, we investigated neural distribution of the CPM to determine the area within the CPM that BoNT could be effectively and safely injected.
      Clear localization of the targeted muscle is an initial step for effective and safe use of BoNT. However, even if the location of the CPM is clarified during BoNT injection using various methods (anatomical landmark, electromyography, or ultrasonography), impairment of vocal fold movement following BoNT injection has been reported [
      • Kelly EA
      • Koszewski IJ
      • Jaradeh SS
      • Merati AL
      • Blumin JH
      • Bock JM
      Botulinum toxin injection for the treatment of upper esophageal sphincter dysfunction.
      ,
      • Kim DY
      • Park CI
      • Ohn SH
      • Monn JY
      • Chang WH
      • Park SW.
      Botulinum toxin type A for poststroke cricopharyngeal muscle dysfunction.
      . The upper zone of the lateral area of the CPM (Zone 1) is adjacent to the PCAM the most. The PCAM is the only abductor muscle among intralaryngeal muscles. Since BoNT can spread to the adjacent muscle through a fascia [
      • Ramirez-Castaneda J
      • Jankovic J
      • Comella C
      • Dashtipour K
      • Fernadez HH
      • Mari Z.
      Diffusion, spread, and migration of botulinum toxin.
      ], we should pay attention when injecting BoNT along the upper margin of the cricoid cartilage to minimize the risk of local diffusion of BoNT to the fascia of the PCAM, especially in zone 1. Axonal transport of BoNT can cause complications [
      • Ramirez-Castaneda J
      • Jankovic J
      • Comella C
      • Dashtipour K
      • Fernadez HH
      • Mari Z.
      Diffusion, spread, and migration of botulinum toxin.
      ,
      • Bomba-Warczak E
      • Vevea JD
      • Brittain JM
      • Figueroa-Bernier A
      • Tepp WH
      • Johnson EA
      • et al.
      Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.
      . Therefore, injecting BoNT along the lower margin of the cricoid cartilage should also be careful to minimize the possibility of axonal diffusion, especially in Zone 2 where the nerve ending of the EBRLN is found in some specimens. Therefore, large dose injection of BoNT at the lateral area of the CPM should be cautious to minimize the risk of impairing vocal fold movement. Since injected BoNT is distributed within the muscle tissue in the shape of a long and thin layer, parallel to the muscle fiber [
      • Elwischger K
      • Kasprian G
      • Weber M
      • Meyerspeer M
      • Linder C
      • Auff E
      • et al.
      Intramuscular distribution of botulinum toxin–visualized by MRI.
      ], the best way to perform BoNT injection via a transcutaneous approach is by performing needle insertion through the middle height of the cricoid cartilage when BoNT is injected at the lateral area of the CPM.
      Although a large dose of BoNT injection can lead to blockage of acetylcholine release at the neuromuscular junction over a wide range of areas, it increases the distance of local diffusion and axonal transport, which can cause unexpected complications [
      • Ramirez-Castaneda J
      • Jankovic J
      • Comella C
      • Dashtipour K
      • Fernadez HH
      • Mari Z.
      Diffusion, spread, and migration of botulinum toxin.
      ,
      • Yi KH
      • Lee HJ
      • Choi YJ
      • Lee JH
      • Hu KS
      • Kim HJ.
      Intramuscular Neural Distribution of Rhomboid Muscles: Evaluation for Botulinum Toxin Injection Using Modified Sihler's Method.
      . Therefore, a minimal but effective dose of BoNT should be injected to avoid unexpected complications. BoNT injection into the area where neuromuscular junction or nerve ending is abundant in the target muscle enables an effective use of BoNT with a minimal dose. An abundant area of the neuromuscular junction is represented as the area where the gross nerve ending or neural arborization pattern is observed on Sihler's staining [
      • Yi KH
      • Lee HJ
      • Choi YJ
      • Lee JH
      • Hu KS
      • Kim HJ.
      Intramuscular Neural Distribution of Rhomboid Muscles: Evaluation for Botulinum Toxin Injection Using Modified Sihler's Method.
      ]. In the present study, the nerve ending or neural arborization pattern originated from the pharyngeal plexus was the most commonly found one in the posterolateral area that was accessible via both transcutaneous and transluminal approaches. Thus, posterolateral area (zone 3 and zone 4) is expected to be a relatively ideal point for BoNT injection with a minimal dose. Neural distribution at the posterior area of the CPM only accessible via a transluminal approach is absent. Therefore, the posterior area is thought to be effective only by local diffusion after BoNT injection. A large dose of BoNT is required to expect a paralytic effect of the CPM when BoNT is injected within the posterior area.

      5. Conclusion

      Although the number of specimens evaluated in the study was small, our results suggested technical considerations according to approaches for injecting BoNT into the CPM. In particular, posterolateral area (zone 3 and zone 4) is thought to be the most suitable area for alleviating spasticity of the CPM with a minimum dose of BoNT. The lateral area of the CPM is easily accessible when using transcutaneous injection. A large dose injection of BoNT at the lateral area needs caution because this area is adjacent to the PCAM with the presence of nerve ending of EBRLN, which can cause impairment of vocal fold movement. In addition, although the posterior area of the CPM can be easily exposed when performing a transluminal approach, this area is hard to expect an effect of BoNT with a small dose.

      Funding

      This work was supported by a grant (NRF-2019R1C1C1010776) of the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP).

      Declaration of Competing Interest

      The authors have no potential conflicts or competing interests relevant to this study to disclose.

      Acknowledgment

      The authors of this study are grateful to donors of cadavers and bereaved families. Bo Hae Kim and Hee-Jin Kim contributed equally to this report as corresponding authors.

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