European Journal of Cardiovascular Medicine

The deep cervical muscles play a pivotal role in the biomechanics of the neck, contributing to the stability and fine control necessary for head and neck movements. These muscles are involved in postural support, especially in static and dynamic position, in regard to small changes in the head position, and in the stabilization of the cervical vertebrae. However, the basic aspects of neurophysiological control of these muscles such as compartmentalisation of the muscles and distribution of muscle spindles within the muscles has received relatively less attention [1]. Muscle spindles are specialised end organs that has the capacity to respond to changes in muscle length and, as a result, offer afferent feedback to the CNS for proprioception and reflexive movements. In so far as cervical muscles are concerned, spindles appear to be particularly important as receptors involved in head and neck position sense or position sense induced by external forces or voluntary movements [2]. These, however, are not located uniformly throughout the muscles; some areas of the muscles are known as spindle rich areas that contain a relatively high number of these receptors. A proper identification of the positions and extents of these areas in the deep cervical muscles is important for studying the pattern of sensory-motor interface pertaining to neck function [3].

Though histological sections or muscle dissection offer some understanding about the muscle and its Neuromuscular relations and structure, these methods are very ineffective to study about the specific patterns of innervations and the location of spindle within muscles [4]. Sihler’s staining, a relatively new staining procedure designed for depicting the pattern of muscle innervation while preserving the fibers architecture of the muscle, has come out as one of the powerful resources by which study of the intricate fascicular arrangement of nerves within the muscle can be done efficiently [5]. This method involves making the muscle tissue almost translucent by running it through various baths and then staining only the neural elements so that one is able to look at the whole nerve supply and the muscle spindle in the muscle in question [6]. Sihler’s staining has been applied to the deep cervical muscles allowing accurate identification of their neuromuscular compartments. These compartments are differentiated through area within a muscle that has a disparate innervation, which, in most occasions, underlines disparate work and biomechanical necessity [7]. This way, when these compartments are defined, the researchers are able to understand the role that various regions of a muscle play in functional muscle capacity and the manner in which these regions interrelate during various type of movements [8].

Moreover, it also permits specific mapping of the location of muscle spindles in these compartments, that are most densely innervated. This localization is of special interest in the deep cervical muscles in which the areas containing spindles are presumably essential for the delicate regulation of movements within the neck and for the head stability [9]. Knowledge of the topographical location of these muscle spindles could be very useful clinically in view of the role they play in neck pain and other injuries such as whiplash and cervical dislocation [10]. Studies employing Sihler’s staining in the recent past have finally opened up the field of understanding the fibre type organisation of several muscles with more detail; however, relatively very little is known about applying this technique to the deep cervical muscles [11]. Using this approach, the present study intends to make up the existing literature by presenting a detailed account of the NM compartments and spindle disposition in these muscles. These results should therefore improve the current knowledge with regard to functional anatomy of the cervical spine and help in the design of specific therapeutic procedures for disorders of the cervical region of the spine [12].

Objective

To investigate the neuromuscular compartments and identify the center of the highest region of muscle spindles abundance in deep cervical muscles.

This descriptive study was conducted at Navodaya Medical College hospital and Research Centre. Muscle samples were obtained from 33 patients who underwent various surgical procedures in which deep cervical muscles were accessible and could be safely obtained. These muscles were chosen: sternocleidomastoid (SCM) and trapezius (TPZ), while the reasons for selecting them are as follows: The SCM and TPZ muscles are very important in neck movements and their structure is rather complicated. The collected samples were then preserved in a solution as soon as they were collected. Sihler’s staining technique was used to make the muscle tissue transparent by leaving it unstained but stained the nerves and muscle spindles. Muscle samples were initially demineralised by immersing the tissue in a 4% KOH solution in order to soften and depigment the muscle while not destroying the neural structures. To this effect, decalcification was done using a solution of acetic acid to prevent the interference of calcified structures in the muscle with the transparency of the tissue. In order to obtain the better transparency of the samples, bleaching of the samples was done by hydrogen peroxide. This was done to negates the pigmentation as well as to further enjoy the clearing of the muscle tissue. The transparent samples were later treated with Sihler’s staining solution that stained differentially the neural tissues in the muscle. This solution is often composed of hematoxylin and other agents that facilitates adherence on a neural tissue to the extent that they may be viewed through the lens of a microscope. Following staining, the samples were washed with water to remove excess stained before doing neutralization to fix the stained tissue for analysis. Cross sections of the muscle that was stained and cleared were placed on microscopic slides. These specimens were then treated with glycerin so that the specimens do not deteriorate and a transparency of the slides was maintained.

Analysis and Mapping

Once the staining process was complete, the specimens were examined under a light microscope to identify and map the distribution of muscle spindles within the deep cervical muscles. The analysis focused on two primary muscles: the SCM and TPZ. The stained samples were analyzed to identify distinct neuromuscular compartments within the muscles. These compartments were defined by regions of concentrated neural elements, including motor endplates and intramuscular nerves, which were clearly visible due to the Sihler’s staining.

Statistical Analysis

Descriptive statistics, including mean spindle density and standard deviations, were calculated. Comparative analyses were performed between different regions within the SCM and TPZ to assess the variation in spindle density across these muscles

Data were collected from 33 patients. Middle third of the SCM muscle exhibits the highest density of motor endplates, averaging 12 ± 3 per square centimeter, suggesting its role in fine motor control. The upper third of the SCM has a moderate density with 6 ± 2 motor endplates/cm², while the lower third shows the lowest density at 4 ± 1 motor endplates/cm². In the TPZ muscle, the middle region also has the highest motor endplate density at 10 ± 2 per square centimeter, followed by the upper region with 8 ± 2, and the lower region with 5 ± 1 motor endplates/cm², indicating a similar pattern of neuromuscular organization.

Table 1: Neuromuscular Compartmentalization in SCM and TPZ Muscles

The results show that the middle third of the SCM muscle has the highest spindle density, averaging 15 ± 3 spindles per square centimeter, indicating its critical role in proprioception and fine motor control. The upper third of the SCM has a lower density with 5 ± 1 spindles/cm², while the lower third shows a moderate density of 7 ± 2 spindles/cm². In the TPZ muscle, the middle region has the highest spindle density at 8 ± 2 spindles per square centimeter, followed by the upper region with 6 ± 2, and the lower region with 4 ± 1 spindles/cm², reflecting its supportive role in broader, less precise movements.

Table 2: Muscle Spindle Density in SCM and TPZ Muscles

The middle third of the SCM exhibits a significantly higher spindle density (15 ± 3 spindles/cm²) compared to the TPZ's middle region (8 ± 2 spindles/cm²), with a p-value of 0.001, indicating high statistical significance. In the lower third/region, the SCM also has a higher spindle density (7 ± 2 spindles/cm²) than the TPZ (4 ± 1 spindles/cm²), with a p-value of 0.03, denoting statistical significance. However, the difference in spindle density between the upper third of the SCM and the upper region of the TPZ is not statistically significant (p = 0.08).

Table 3: Comparative Analysis of Spindle Density Between SCM and TPZ Muscles

* p < 0.05, statistically significant ** p < 0.01, highly significant

In the SCM muscle, the middle third shows the highest correlation coefficient (r = 0.92), indicating a very strong relationship between motor endplate density (12 per cm²) and spindle density (15 per cm²). The upper third of the SCM also demonstrates a strong correlation (r = 0.85) with 6 motor endplates and 5 spindles per cm², while the lower third shows a moderately strong correlation (r = 0.78). Similarly, in the TPZ muscle, the middle region has a high correlation coefficient (r = 0.87) between motor endplate density (10 per cm²) and spindle density (8 per cm²), followed by the upper region (r = 0.81) and the lower region (r = 0.79).

Table 4: Correlation Between Motor Endplate Density and Muscle Spindle Density

The findings of this study, utilizing Sihler’s staining technique to map the neuromuscular compartments and muscle spindle distribution in the deep cervical musculature, provide significant insights into the functional anatomy of the sternocleidomastoid (SCM) and trapezius (TPZ) muscles [12]. We found from our study that pedicle and vertebral muscles to be well organized and propose certain roles that distinct areas within these muscles play in cervical movements and position sense. Division of neuromuscular compartments in the SCM and TPZ muscles has given a new insight into the manner in which these muscles had been recruited during head and neck movements [13]. The portion of the SCM, especially the middle third showed the highest concentration of ME and MS. Such compartmentalization tells about stratified organization in the SCM, in which various segments are oriented toward differentiated degrees of motor control. This middle third contains a dense innervation and high spindle density for the musculature, and is probably highly relevant to head positioning and proprioceptive feedback, which are required for stability and coordinated movements [14].

However, the TPZ muscle was more structureless with irregular neuromuscular elements and the densities in the middle part of the muscle was slightly higher. As for the present findings, this pattern points towards a function that is more suitable for playing a part in other actions that are less specific and more general in nature, particularly those involving the shoulder girdle and stability of the scapula [15]. Based on these results, one might assume that the role of the TPZ is retained more in terms of postural control and axial movements of the gross motor patterns and rather does not seem to figure in fine motor control, thus clearly correlating with muscle attachment origin and the involvement of the scapula [16]. It is evident from this study that the organization of Muscle spindles in the SCM and TPZ implies the functional sub division within these muscles. These set of observations we believe points to the middle third of the SCM being specialized for proprioceptive function with an average spindle density of 15 spindles per square centimeter. This specialization is particularly important for all those assignments, which involve high degree of motor coordination as the head stabilization and accurate positioning in the response to the stimuli [17].

Muscle spindles were also found in the TPZ and they had a lesser density of distribution as compared to the rest of the zones, and the density was also uniform with slightly higher spindle density in the middle region of the TPZ in comparison to either the upper or the lower region. Such distribution pattern of the TPZ is also consistent with the TPZ’s involvement in coarser, and lower level movement functions of the human body such as arm and shoulder movements [18]. The count of spindle density in the TPZ is lesser than the SCM even though the latter is involved in fine cervical movements while the former is involved in shoulder stability. Motor endplate density and muscle spindles density were significantly correlated with correlation coefficient ranging from 0. 78 to 92 in both the SCM and TPZ muscles indicating the motor control and sensory feedback is closely linked in these muscles. Muscle areas with higher motor endplate densities had a higher spindle density as well, suggesting that parts of the muscle for delicate motor co-ordination have better proprioceptive feedback systems [19]. This relationship shows that motor and sensory activity are mutually interconnected when it comes to the cervical muscles as far as detailed coordination and balance of precise movements are concerned [20]. The additional anatomic description of great detail of compartments in the deep cervical muscles, location of muscle spindles can be of paramount clinical relevance in the treatment of cervical pain syndromes, whiplash injuries and other cervical musculoskeletal disorders.

It is concluded that the sternocleidomastoid (SCM) and trapezius (TPZ) muscles exhibit distinct neuromuscular compartmentalization and muscle spindle distribution, with the middle third of the SCM showing the highest density of spindles, crucial for fine motor control and proprioception. These findings have important implications for understanding cervical muscle function and developing targeted rehabilitation strategies for neck disorders

1. Wang D, Chen P, Jia F, Wang M, Wu J, Yang S. Division of neuromuscular compartments and localization of the center of the highest region of muscle spindles abundance in deep cervical muscles based on Sihler's staining. Front Neuroanat. 2024 May 22;18:1340468. doi: 10.3389/fnana.2024.1340468. PMID: 38840810; PMCID: PMC11151460.
2. Hu X., Wang M., He X., Chen P., Jia F., Wang D., et al. (2023). Division of neuromuscular compartments and localization of the center of the intramuscular nerve-dense region in pelvic wall muscles based on Sihler's staining. Anat. Sci. Int. 99, 127–137. doi: 10.1007/s12565-023-00744-4
3. Kurtys K., Podgórski M., Gonera B., Vazquez T., Olewnik Ł. (2023). An assessment of the variation of the intramuscular innervation of the gracilis muscle, with the aim of determining its neuromuscular compartments. J. Anat. 242, 354–361. doi: 10.1111/joa.13785
4. Yu J., Li Y., Yang L., Li Y., Zhang S., Yang S. (2023). The highest region of muscle spindle abundance should be the optimal target of botulinum toxin A injection to block muscle spasms in rats. Front. Neurol. 14:1061849. doi: 10.3389/fneur.2023.1061849
5. Gilcrease-Garcia B. M., Deshmukh S. D., Parsons M. S. (2020). Anatomy, imaging, and pathologic conditions of the brachial plexus. Radiographics 40, 1686–1714. doi: 10.1148/rg.2020200012
6. Wang J., Wang Q., Zhu D., Jiang Y., Yang S. (2020). Localization of the center of the intramuscular nerve dense region of the medial femoral muscles and the significance for blocking spasticity. Ann. Anat. 231:151529. doi: 10.1016/j.aanat.2020.151529
7. Farrell M., Karp B. I., Kassavetis P., Berrigan W., Yonter S., Ehrlich D., et al. (2020). Management of Anterocapitis and Anterocollis: a novel ultrasound guided approach combined with electromyography for botulinum toxin injection of longus Colli and longus capitis. Toxins (Basel) 12:626. doi: 10.3390/toxins12100626
8. Gross J. H., Zenga J., Sharon J. D., Jackson R. S., Pipkorn P. (2019). Longus capitis reconstruction of the soft palate. Otolaryngol. Head Neck Surg. 161, 536–538. doi: 10.1177/0194599819849031
9. Castagna A., Albanese A. (2019). Management of cervical dystonia with botulinum neurotoxins and EMG/ultrasound guidance. Neurol Clin Pract. 9, 64–73. doi: 10.1212/CPJ.0000000000000568
10. Jones M. R., Prabhakar A., Viswanath O., Urits I., Green J. B., Kendrick J. B., et al. (2019). Thoracic outlet syndrome: a comprehensive review of pathophysiology, diagnosis, and treatment. Pain Ther. 8, 5–18. doi: 10.1007/s40122-019-0124-2
11. Zhang X. Y., Ma T. T., Liu L., Yin N. B., Zhao Z. M. (2017). Anatomic study of the musculus longus capitis flap. Surg. Radiol. Anat. 39, 271–279. doi: 10.1007/s00276-016-1708-8
12. Allison S. K., Odderson I. R. (2016). Ultrasound and electromyography guidance for injection of the longus Colli with botulinum toxin for the treatment of cervical dystonia. Ultrasound Q. 32, 302–306. doi: 10.1097/RUQ.0000000000000226
13. Isner-Horobeti M. E., Muff G., Lonsdorfer-Wolf E., Deffinis C., Masat J., Favret F., et al. (2016). Use of botulinum toxin type a in symptomatic accessory soleus muscle: first five cases. Scand. J. Med. Sci. Sports 26, 1373–1378. doi: 10.1111/sms.12616
14. Reichel G., Stenner A., von Sanden H., Herrmann L., Feja C., Löffler S. (2016). Endoscopic-guided injection of botulinum toxin into the longus capitis muscle and into the obliquus superior part of the longus colli muscle in dystonic antecaput: our experience. Basal Ganglia 6, 97–100. doi: 10.1016/j.baga.2016.01.007
15. Simpson D. M., Hallett M., Ashman E. J., Comella C. L., Green M. W., Gronseth G. S., et al. (2016). Practice guideline update summary: botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: report of the guideline development Subcommittee of the American Academy of neurology. Neurology 86, 1818–1826. doi: 10.1212/WNL.0000000000002560
16. Wang D, Chen P, Jia F, Wang M, Wu J, Yang S. Division of neuromuscular compartments and localization of the center of the highest region of muscle spindles abundance in deep cervical muscles based on Sihler's staining. Front Neuroanat. 2024 May 22;18:1340468. doi: 10.3389/fnana.2024.1340468. PMID: 38840810; PMCID: PMC11151460.
17. Sun Y, Fede C, Zhao X, Del Felice A, Pirri C, Stecco C. Quantity and Distribution of Muscle Spindles in Animal and Human Muscles. International Journal of Molecular Sciences. 2024; 25(13):7320. https://doi.org/10.3390/ijms25137320
18. Wang, D.; Chen, P.; Jia, F.; Wang, M.; Wu, J.; Yang, S. Division of neuromuscular compartments and localization of the center of the highest region of muscle spindles abundance in deep cervical muscles based on Sihlers staining. Front. Neuroanat. 2024, 18, 1340468.
19. hou, J.; Jia, F.; Chen, P.; Zhou, G.; Wang, M.; Wu, J.; Yang, S. Localisation of the centre of the highest region of muscle spindle abundance of anterior forearm muscles. J. Anat. 2024, 244, 803–814.
20. Brandl, A.; Egner, C.; Reer, R.; Schmidt, T.; Schleip, R. Associations between Deformation of the Thoracolumbar Fascia and Activation of the Erector Spinae and Multifidus Muscle in Patients with Acute Low Back Pain and Healthy Controls: A Matched Pair Case-Control Study. Life 2022, 12, 1735