Re: 표재성 근막의 신경지배 2022 논문 탐구!!

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Front Neuroanat

. 2022 Aug 29;16:981426. doi: 10.3389/fnana.2022.981426

Innervation of human superficial fascia

Caterina Fede 1,*, Lucia Petrelli 1, Carmelo Pirri 1, Winfried Neuhuber 2, Cesare Tiengo 3, Carlo Biz 4, Raffaele De Caro 1, Robert Schleip 5, Carla Stecco 1

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PMCID: PMC9464976  PMID: 36106154

Abstract

The superficial fascia has only recently been recognized as a specific anatomical structure. Furthermore, whereas it is actually recognized that the innervation of the deep/muscular fascia plays a key role in proprioception and nociception, there are very few studies that have analyzed these characteristics in the superficial fascia. In this work, our group analyzed two different anatomical districts (abdomen and thigh), from volunteer patients, undergoing surgery procedures. Each sample was processed for histological analysis by Hematoxylin&Eosin, and by immunohistochemistry stainings (in 5-micron-paraffin embedded section and in cryosectioned free floating samples), with antibodies specific for nerve fibers: S100 antibody for myelinating and non-myelinating Schwann cells, PGP9.5 antibody as pan-neuronal marker, tyrosine hydroxylase for autonomic innervation. The results revealed a huge innervation: the nervous structures were found above all around blood vessels and close to adipocytes, but they penetrated also in the connective tissue itself and are found in the midst of fibro-adipose tissue. The tissue is pervaded by both thin (mean diameter of 4.8 ± 2.6 μm) and large nerve fiber bundles of greater diameter (21.1 ± 12.2 μm). The ratio S100/TH positivity was equal to 2.96, with a relative percentage of autonomic innervation with of 33.82%. In the light of these findings is evident that the superficial fasciae have a clear and distinct anatomical identity and a specific innervation, which should be considered to better understand their role in thermoregulation, exteroception and pain perception. The knowledge of the superficial fascia may improve grading and developing of different manual approach for treatments of fascial dysfunctions, and the understanding of how some factors like temperature or manual therapies can have an impact on sensitivity of the fascia.

Abstract

표재성 근막은 

최근에야 특정 해부학적 구조로 인식되기 시작했습니다. 

또한 

심부/근막의 신경 분포가 

고유 수용성 감각과 통각에 중요한 역할을 한다는 사실은 알려져 있지만, 

표재성 근막의 이러한 특성을 분석한 연구는 거의 없습니다. 

이 연구에서 우리 그룹은 

수술 절차를 거친 자원 환자의 

두 가지 해부학적 부위(복부 및 허벅지)를 분석했습니다. 

각 샘플은 신경 섬유에 특이적인 항체를 사용하여 헤마톡실린&에오신과 면역조직화학 염색(5마이크론 파라핀 매립 절편 및 냉동 절편 자유 부유 샘플)을 통해 조직학적 분석을 위해 처리되었습니다: 수초성 및 비수초성 슈반 세포에 대한 S100 항체, 범신경 세포 마커로서 PGP9.5 항체, 자율 신경 분포에 대한 티로신 하이드 록실 라제. 

그 결과 

신경 구조는 무엇보다도 혈관 주변과 지방 세포 근처에서 발견되었지만 

결합 조직 자체에도 침투하여 섬

유 지방 조직 한가운데에서도 발견되는 등 

거대한 신경 분포가 밝혀졌습니다. 

조직에는 얇은(평균 직경 4.8 ± 2.6 μm) 신경 섬유 다발과 

더 큰 직경(21.1 ± 12.2 μm)의 큰 신경 섬유 다발이 모두 퍼져 있습니다. S100/TH 양성 비율은 2.96이었으며, 

자율 신경 분포의 상대적 비율은 33.82%였습니다. 

이러한 결과에 비추어 볼 때 

표재성 근막은 명확하고 뚜렷한 해부학적 정체성과 특정 신경 분포가 있으며

체온 조절, 외부 감각 및 통증 지각에서 그 역할을 더 잘 이해하기 위해

고려되어야한다는 것이 분명합니다.

표재성 근막에 대한 지식은

근막 기능 장애 치료를 위한 다양한 수기 접근법의 등급 지정 및 개발, 온도 또는 수기 치료와 같은

일부 요인이 근막의 민감도에 미치는 영향에 대한 이해를 향상시킬 수 있습니다.

Keywords: superficial fascia, hypodermis, innervation, nerve fibers, autonomic innervation

Introduction

The superficial fascia has only recently been recognized as a specific anatomical structure in its own right, being originally considered as included in the hypodermis. It consists of a fibrotic layer in the middle of hypodermis, dividing the superficial adipose tissue (SAT) from the deep adipose tissue (DAT), which corresponds to the membranous layer of the tela subcutanea of the Terminologia Anatomica (Abu-Hijleh et al., 2006, FIPAT). The drastic growth of articles published on the subcutaneous tissue and the superficial fascia dates back to the last 15–20 years. Furthermore, it is mainly the fields of plastic surgery and radiology that take into consideration the superficial fascia and distinguish it as a single anatomical entity: of about 2,000 articles mentioning the superficial fascia, as many as 30% refer to surgery and 10% to radiology.

Many authors emphasize the importance of an intimate knowledge of the superficial fascia system for both reconstructive and cosmetic breast surgery (Rehnke et al., 2018), and for abdominoplasty procedures (Koller and Hintringer, 2012). Song et al. (2006) demonstrated by an ex vivo model that the maintenance of the biomechanical properties of the superficial fascia during surgery procedures is fundamental to enhance surgical outcome through anchoring of deeper tissues. Radiologists on the other hand point out that Magnetic Resonance Imaging (MRI) is a very useful imaging technique to detect localized fascial involvement and assess its extent in trauma, infections and also neoplastic diseases (Kirchgesner et al., 2019). MRI permits also the eval‎uation of various forms of lymphedema, highlighting the water retention diffusely spread over the entire dermis, and an important fluid retention located in the interlobular spacing and beside the superficial fascia, together with an increase of the mean thickness of the superficial fat lobules (Idy-Peretti et al., 1998).

Until now there are very few studies that have analyzed the amount and type of innervation present in the superficial fascia. Some studies refer to the innervation of the hypodermis in general: Chi et al. (2021) demonstrated a dense sympathetic innervation in the subcutaneous fat, whereas Glatte described that post-ganglionic C-fibers of the autonomic nervous system control the thermoregulation of the subdermal plexus at the interface between hypodermis and dermis (Glatte et al., 2019). Recently, it was estimated that the entire fascial network in the human body counts approximately 250 million of nerve endings (Schleip, 2020). This leads to consider the fascia as the richest sensory tissue of the human body, drawing the attention of surgeons and clinicians.

Recently, our group (Fede et al., 2020), analyzing the various soft tissues of the human hip region, demonstrated that the superficial fascia was the second most highly innervated tissue (with a mean density of nerves of 33.0 ± 2.5/cm2) after the skin (64.0 ± 5.2/cm2). Also SAT (superficial adipose tissue) and DAT (deep adipose tissue) were less innervated with respect to the fascia, with a mean density of innervation equal to 14.5 ± 1.6/cm2 and 15.0 ± 6.3/cm2, respectively. That work, however, was only a quantitative analysis performed by a general marker (S100 antibody), that does not allow to distinguish the autonomic or sensory innervation.

Thus, in the present work, the innervation of the human superficial fascia of the abdomen and of the hip region were studied to identify the presence of neural structures and their distribution, with a particular focus to the autonomic nervous structures. An improved knowledge of the fascial tissue innervation may lead to better understanding of the sensitivity of the superficial fascia, and may help to demonstrate how some factors like temperature or manual therapies can have an impact on some dysfunctions related to the superficial fascia.

소개

표재성 근막은 원래 피하에 포함된 것으로 여겨지다가 최

근에야 그 자체로

특정 해부학적 구조로 인식되기 시작했습니다.

표피 근막은

표피 지방 조직(SAT)과 심부 지방 조직(DAT)을 구분하는

피하지방 중앙의 섬유층으로 구성되어 있으며,

이는 용어해부학 용어집의 피하 막층에 해당합니다(Abu-Hijleh 외., 2006, FIPAT).

The superficial fascia has only recently been recognized as a specific anatomical structure in its own right, being originally considered as included in the hypodermis. It consists of a fibrotic layer in the middle of hypodermis, dividing the superficial adipose tissue (SAT) from the deep adipose tissue (DAT), which corresponds to the membranous layer of the tela subcutanea of the Terminologia Anatomica (Abu-Hijleh et al., 2006, FIPAT)

피하 조직과 표재성 근막에 관한 논문의 급격한 성장은

지난 15~20년으로 거슬러 올라갑니다.

또한 표재성 근막을 고려하고 이를 하나의 해부학적 실체로 구분하는 것은

주로 성형외과와 영상의학 분야입니다.

표재성 근막을 언급한 약 2,000개의 논문 중 30%는 외과, 10%는 영상의학에 관한 것입니다.

많은 저자들은 재

건 및 미용 유방 수술(Rehnke et al., 2018)과 복부 성형술(Koller and Hintringer, 2012)에 있어

표재성 근막 시스템에 대한 친밀한 지식이 중요하다고 강조합니다.

Song 등(2006)은 생체 외 모델을 통해

수술 과정에서

표재성 근막의 생체 역학적 특성을 유지하는 것이

심부 조직의 고정을 통해 수술 결과를 향상시키는 데 필수적이라는 것을 입증했습니다.

반면에 방사선 전문의들은

자기공명영상(MRI)이 외상, 감염 및 종양성 질환에서 국소적인 근막 침범을 감지하고

그 정도를 평가하는 데 매우 유용한 영상 기술이라고 지적합니다(Kirchgesner 등., 2019).

MRI는 또한 다양한 형태의 림프부종을 평가할 수 있으며,

전체 진피에 확산되어 있는 수분 저류와 소엽 간 간격 및 표면 근막 옆에 위치한 중요한 체액 저류와 함께 표면 지방 소엽의 평균 두께의 증가를 강조합니다(Idy-Peretti et al., 1998).

지금까지

표재성 근막에 존재하는 신경 분포의 양과 유형을 분석한 연구는

거의 없습니다.

일부 연구는 일반적으로 표피의 신경 분포에 대해 언급합니다:

Chi 등(2021)은

피하지방에

교감신경이 밀집되어 있음을 입증한 반면,

Glatte는

자율신경계의 신경절 후 C 섬유가 피하와 진피 사이의 경계에서

피하 신경총의 체온 조절을 제어한다고 설명했습니다(Glatte 등, 2019).

최근 인체의 전체 근막 네트워크에는

약 2억 5천만 개의 신경 종말이 있는 것으로 추정되었습니다(Schleip, 2020).

이로 인해

근막은

인체에서 가장 풍부한 감각 조직으로 간주되어

외과의와 임상의의 관심을 끌고 있습니다.

최근 우리 그룹(Fede 등, 2020)은

인간 고관절 부위의 다양한 연조직을 분석한 결과,

표재성 근막은 피부(64.0 ± 5.2/cm2) 다음으로

신경 밀도가 높은 조직(평균 신경 밀도 33.0 ± 2.5/cm2)임을 입증했습니다.

또한

SAT(표재성 지방 조직)와 DAT(심부 지방 조직)는

근막에 비해 신경 밀도가 낮았으며,

평균 신경 밀도는 각각 14.5 ± 1.6/cm2와 15.0 ± 6.3/cm2로 나타났습니다.

그러나

이 연구는 일반적인 마커(S100 항체)를 이용한 정량적 분석에 불과하여

자율신경과 감각 신경을 구분할 수 없었습니다.

따라서

본 연구에서는

복부 및 고관절 부위의 인간 표면 근막의 신경 분포를 연구하여

자율 신경 구조에 특히 중점을 두고

신경 구조의 존재와 그 분포를 확인했습니다.

근막 조직 신경 분포에 대한 지식이 향상되면

표재성 근막의 민감도를 더 잘 이해할 수 있으며,

온도나 수기 요법과 같은 일부 요인이

표재성 근막과 관련된 일부 기능 장애에 어떤 영향을 미칠 수 있는지 설명하는 데

도움이 될 수 있습니다.

Materials and Methods

Samples collection

The Superficial Fascia (SF, 700 μm thickness, Pirri et al., 2022) was collected in the abdominal region (from three volunteer patients who were undergoing abdominoplasty at the Plastic and Reconstructive Surgery Unit of the University of Padua), and in the hip area (from six volunteer patients undergoing elective surgical procedures at the Orthopaedic Clinic of the University of Padua).

The ethical regulations regarding research on human tissues were carefully followed (approval no. 3722/AO/16, study approved on 21 April 2016 by the Ethical Committee for clinical trials in the province of Padova). The research was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki as revised in 2000 and those of Good Clinical Practice. All subjects participating in the study received a thorough explanation of the risks and benefits of inclusion and gave their oral and written informed consent to publish the data.

All the samples were at least 0.5 cm × 1.5 cm, and derived from adults: two females/one male, age between 37 and 45, in the hip area, and three females/three males, age >55, in the abdomen region.

Samples of superficial fascia from the hip region were all formalin-fixed (in 10% buffered formaldehyde, pH 7.4) for immunohistochemistry analysis and histological eval‎uation.

In the abdominal region, given the large area where the samples are taken, 8–10 specimens per subject were taken in different areas of the SF, in about 40 cm2 of total area, in full thickness (from skin to SF). One specimen per subject was eval‎uated in full thickness by hematoxylin and eosin stain, in the others the SF was isolated and cleansed of superficial and deep adipose tissue.

The isolated SF from abdomen, fixed in 10% buffered formaldehyde, pH 7.4, were divided in two groups: three SF (for each patient) were used for floating immunohistochemistry, the other 5–7 SF followed the protocol for classical paraffin embedding.

For floating immunohistochemistry, the SF samples were washed in Phosphate Buffered Saline (PBS) and transferred in water with increasing series of cryo-preservative solution (sucrose 10%–20%–30% w/v, 5 days for each solution), and then were frozen in Isopentane (2-Methylbutane-Merck) cooled at −80°C in ice dry. Sections of 60 μm were cut at −20°C in a Cryostat (Leica—CM1850), then washed in PBS and processed for floating Immunohistochemistry (see paragraph below).

The other fixed samples were dehydrated in graded ethanol and in xylene, and embedded in paraffin. Five-μm tangential sections were cut by microtome, dewaxed and hydrated for Hematoxylin and Eosin stain (Figure 1D), to eval‎uate the general morphology and organization of the tissue, and for Immunohistochemical protocol, as detailed in the paragraph below.

Figure 1.

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Superficial fascia layer of the abdomen. (A) Formalin-fixed sample of the abdominal region, from skin (E: epidermis, D: dermis) to the superficial fascia (SF). (B) Hematoxylin and eosin stain of all the layers from skin (E: epidermis, D: dermis), to the deep adipose tissue (DAT). The superficial fascia (SF) is the fibrous layer localized between the superficial adipose tissue (SAT, organized in lobules separated by the fibrous septa of the retinacula cutis, RC) and the deep adipose tissue (DAT). The SF highlighted in (A) is isolated from the SAT and the DAT (C) and stained by Hematoxylin and Eosin (D). Panel (D) is a tangential section of the flat embedded SF as in (C). Histologically the SF layer if formed by a net of collagen fibers arranged irregularly (c: connective tissue), interconnected and mixed with adipocytes (a) and crossed by blood vessels (v). Scale Bars = 3 mm.

Immunohistochemistry

Dewaxed sections and floating samples were treated with blocking of endogenous peroxidases (1% H2O2 in PBS), incubated in blocking solution [PBS + 0.2% bovine serum albumin (BSA)] for 1 h and then incubated overnight at 4°C with Rabbit Polyclonal Anti S100 (marker for myelinating and non-myelinating Schwann cells, Agilent-Dako, dilution 1:4,000), Rabbit Anti Tyrosine Hydroxylase (TH, for autonomic innervation, GeneTex, dilution 1:500), or Rabbit Anti PGP9.5 (as a pan-neuronal marker, Merck Millipore, dilution 1:500). After repeated PBS washing, the samples were incubated with the secondary Goat anti rabbit (Jackson, dilution 1:300) for 1 h. Negative controls were processed with the same protocol with the omission of the primary antibodies. The reaction was then developed with 3,3’-diaminobenzidine (Liquid DAB + substrate Chromogen System kit Dako) and stopped with distilled water. Samples were finally counterstained with hematoxylin.

The images were acquired by using Leica DMR microscope (Leica Microsystems, Wetzlar, Germany).

Image analysis

The image analysis was performed only in the samples collected from abdominoplasty, given the large area and the higher precision of collection, which can permit a more accurate analysis. Images were acquired through Leica DMR (Leica Microsystems, Wetzlar, Germany) and computerized image analysis was performed with ImageJ software in serial sections (at least 20 pictures for each sample, enlargement 10×), to quantify the area (%) positive to Tyrosine Hydoxylase and S100 and to calculate the S100/TH ratio.

In addition, the mean diameter of the nerve fiber bundles was calculated by ImageJ software (enlargement 40×) in the three samples from abdomen, in at least 15 S100-positive images per sample. Recognitions of axons by light microscopy can overestimate the diameter of the nervous fibers, for the possible inaccurate identification of very small fibers: our analysis permitted however the identification of bundles of axons up to 2 μm of diameter.

Results

The full thickness specimen (from skin to SF, Figures 1A,B) highlighted as superficial fascia (SF) divides the superficial adipose tissue (SAT), organized in fat lobules with evident fibrous septa (retinacula cutis superficialis), from the deep adipose tissue (DAT). SF is a well-defined thin fibrous layer (Figure 1C), approximately 500–700 μm thick. The Hematoxylin and Eosin stain (Figure 1D) of the SF demonstrated as it is formed by fibro-fatty connective tissue: the collagen fibers are irregularly arranged, spatially interconnected and mixed with adipocytes, and pervaded by blood vessels of different caliber supplying and crossing the tissue. The same organization was evident also in Figure 2A: collagen fibers form a net, between areas of adipocytes and large vessels. The immunohistochemistry by S100 antibody revealed the presence of a huge innervation in the superficial fascia, above all in the wall of the blood vessels (Figures 2A,B,E) and near the adipocytes (Figures 2A,C), but also in the connective tissue itself (Figures 2A,D). The nervous structures pervading the tissue are both thin nerve fiber bundles (mean diameter of 4.8 ± 2.6 μm) and larger nerve fiber bundles of bigger diameter (21.1 ± 12.2 μm; Figure 2D), with approximately the same distribution.

결과

전체 두께 표본(피부에서 SF까지, 그림 1A,B)은

표면 근막(SF)으로 강조 표시되어 있으며,

섬유질 격막(표면 망막)이 뚜렷한 지방 소엽으로 구성된

표면 지방 조직(SAT)을 심부 지방 조직(DAT)과 구분합니다.

SF는

약 500~700μm 두께의 잘 정의된 얇은 섬유층입니다(그림 1C).

SF의 헤마톡실린 및 에오신 염색(그림 1D)은 섬유-지방 결합 조직에 의해 형성된 것으로 콜라겐 섬유가 불규칙하게 배열되고, 공간적으로 상호 연결되고, 지방세포와 혼합되어 있으며, 조직을 공급하고 교차하는 다양한 구경의 혈관에 의해 퍼져 있음을 보여주었습니다.

그림 2A에서도 지방세포와 큰 혈관 사이에 콜라겐 섬유가 그물망을 형성하는 동일한 조직을 확인할 수 있습니다. S100 항체에 의한 면역 조직 화학은 표재성 근막, 특히 혈관 벽 (그림 2A,B,E)과 지방 세포 근처 (그림 2A,C)뿐만 아니라 결합 조직 자체 (그림 2A,D)에도 거대한 신경 분포가 존재한다는 것을 밝혀 냈습니다. 조직에 퍼져 있는 신경 구조는 얇은 신경 섬유 다발(평균 직경 4.8 ± 2.6 μm)과 더 큰 직경의 큰 신경 섬유 다발(21.1 ± 12.2 μm, 그림 2D) 모두 거의 동일한 분포로 존재합니다.

Figure 2.

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Distribution of the innervation in superficial fascia: immunohistochemistry by S100 antibody. (A) The connective tissue (c) is crossed by blood vessels (v) and interconnected with areas rich in adipocytes (a). Nervous structures (*indicates big nerves, arrowheads indicate thin nerve fibers) are evident between the collagen fibers, near the adipocytes and in the wall of blood vessels. (B) A nerve (*) and thin nerve fibers (arrow) close to two blood vessels (v). (C) Innervation between the adipocytes area. (D) Nerves crossing the fibrous connective tissue of the SF. (E) Innervation of the wall of a blood vessel supplying the SF. a, adipocyte; v, vessel; c, connective tissue; *, nerve. Arrowheads indicate small nerve fibers. Scale Bars: (A) = 200 μm, (B–E) = 50 μm.

The immunohistochemistry analyses, both in paraffine-embedded sections and in free-floating samples, permitted to demonstrate a rich innervation of the superficial fascia. The nerves are particularly concentrated in the blood vessels’ adventitia (Figures 3, 4), but they penetrate also in the connective tissue without obvious vascular relation (Figure 3) and are found in the midst of the fibro-adipose tissue (Figures 5, 6). The innervation can be considered both sensory and autonomic (sympathetic), because nerve fibers reactive for S100 and PGP 9.5, a pan-neuronal marker, outnumber those reactive for TH (Figures 3, 4, 5, and 6). In general, the reaction with PGP 9.5 antibody showed thinner positive fibers less contrasted around the blood vessels (Figures 4C,F) and in the dense connective areas (Figures 5C,F), with some varicosities in the axons. At the same time, there is a huge innervation also in the looser connective areas: this is characterized by wavy collagen fibers, large blood vessels that branch out into the dense tissue, nerves and groups of adipocytes. In that areas some large nerve fascicles (up to 200 μm of diameter) cross the SF, presumably on their way to the skin, and showed positive reactions to all the markers: S100 (Figure 7A), TH (Figure 7B), PGP 9.5 (Figure 7C). TH positive nerve fibers were outnumbered by those immunostained by the general markers S100 and PGP 9.5.

파라핀 매립 절편과 자유 부유 샘플 모두에서 면역 조직 화학 분석을 통해 표재성 근막의 풍부한 신경 분포를 확인할 수 있었습니다. 신경은 특히 혈관의 돌출부에 집중되어 있지만(그림 3, 4), 명백한 혈관 관계 없이 결합 조직에도 침투하며(그림 3), 섬유-지방 조직 한가운데에서도 발견됩니다(그림 5, 6). 감각 신경과 자율 신경(교감 신경)은 모두 감각 신경으로 간주할 수 있는데, 범신경 마커인 S100과 PGP 9.5에 반응하는 신경 섬유가 TH에 반응하는 것보다 많기 때문입니다(그림 3, 4, 5, 6). 일반적으로 PGP 9.5 항체와의 반응은 혈관 주변(그림 4C,F)과 밀집된 결합 부위(그림 5C,F)에서 더 얇은 양성 섬유가 덜 대조적으로 나타났으며 축삭에 약간의 변이가 있었습니다. 동시에 느슨한 결합 부위에도 거대한 신경 분포가 있는데, 이는 물결 모양의 콜라겐 섬유, 치밀한 조직, 신경 및 지방 세포 그룹으로 분기되는 큰 혈관이 특징입니다. 이 부위에서는 일부 큰 신경 근막(최대 직경 200μm)이 SF를 가로질러 피부로 향하는 것으로 추정되며 모든 마커에 대해 긍정적인 반응을 보였습니다: S100 (그림 7A), TH (그림 7B), PGP 9.5 (그림 7C). TH 양성 신경 섬유는 일반 마커인 S100 및 PGP 9.5에 의해 면역 염색된 신경 섬유보다 더 많았습니다.

Figure 3.

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Nerve fibers penetrating the connective tissue. Innervation in a free floating superficial fascia of the abdomen region, by anti-S100 antibody. (A) The wall of the blood vessel (v) is richly innervated, and some nerve fibers enter inside the connective tissue (c) of the SF, as shown in the box, enlarged in (B). Panel (C) shows the same anti-S100 reaction in paraffin-embedded 5 μm section. The arrows indicate the nerve fibers. v, blood vessel; c, connective tissue; a, adipocyte. Scale bars: (A) = 200 μm; (B) = 100 μm; (C) = 50 μm.

Figure 4.

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Innervation of the blood vessels of superficial fascia. Superficial fascia of the abdomen, stained with S100 (A,D), Tyrosine Hydroxylase (B,E) and PGP9.5 (C,F) antibodies. Panels (A,B,C) are paraffin-embedded-5 μm samples, whereas (D,E,F) are free-floating samples. All the pictures show the innervation of blood vessels. In (E,F) the varicose nature of the axons is evident. v, blood vessel; a, adipocyte; arrows indicate the nerve fibers. Scale bars: (A,F) = 100 μm; (B,C) = 50 μm; (D,E) = 200 μm.

Figure 5.

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Innervation of the connective tissue. Superficial fascia of the hip (paraffin-embedded-5 μm section: A,B,C) and of abdomen (free-floating samples: D,E,F) stained with S100 (A,D, respectively), Tyrosine Hydroxylase (B,E) and PGP 9.5 (C,F) antibodies. Scale bars: (A,D,E) = 100 μm; (B,C,F) = 50 μm.

Figure 6.

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Innervation in the adipocyte areas: immunohistochemistry with anti-S100, anti-Tyrosine Hydroxylase, and anti-PGP9.5 antibodies. Superficial fascia of the hip (paraffin-embedded-5 μm section: A,B,C) and of abdomen (free-floating sample: D,E,F) stained with S100 (A,D), Tyrosine Hydroxylase (B,E) and PGP9.5 (C,F) antibodies. Arrowheads indicate single nerve fibers. Scale bars: (A,C–F) = 100 μm, (B) = 50 μm.

Figure 7.

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Nerves fascicles passing through SF. Superficial fascia of the hip (A) and of the abdomen (B,C) showing thick fascicles in the SF tissue, stained with S100 (A), Tyrosine Hydroxylase (B), and PGP9.5 (C) antibodies. Scale bars: (A,C) = 100 μm; (B) = 200 μm.

No corpuscular sensory structures (like Pacini or Ruffini) were found in the SF samples, nether in the hip region nor in the abdomen. The specificity of the immunostaining was demonstrated by the absence of reaction in the negative controls (Figure 8).

Figure 8.

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Negative controls. Superficial fascia of the hip (A, paraffin-embedded-5 μm section) and of the abdomen (B, free-floating sample) with the omission of the primary antibody: the negative reaction confirmed the specificity of the immuno-reactions. Scale bars: 100 μm.

The fraction of area (%) positive to Tyrosine Hydoxylase and S100 was estimated to calculate the S100/TH ratio. The analysis was performed only in the abdomen area given the higher quality of the tissue samples from that area and the consequently more reliable analysis. Given the higher background of PGP 9.5 antibody and the less contrasted reaction, the ratio calculation was performed on S100-stained images and TH-stained images, to have a relative quantification of the autonomic innervation in the SF. The immunohistochemistry showed that the TH positive area (IR area %) is around 0.326%, whereas the S100 positive area is around 0.964%, thus leading to a ratio S100/TH positivity of 2.96.

Discussion

This work is the first histological investigation focused on the human SF innervation, which demonstrates that it is hugely innervated, both with autonomic and sensory nerve fibers, according also to our previous work demonstrating that SF of the hip region was the most highly innervated tissue after the skin (skin>Sup Fascia>Deep Fascia>DAT>SAT; Fede et al., 2020). Only some large nerve fascicles cross the superficial fascia to reach the skin, the majority of nerve fibers are located around the vessels, innervating their wall, or are embedded in the fibro-adipose tissue. The perivascular innervation displays a plexus structure in the SF. The floating immunohistochemistry was very helpful to highlight the nervous fibers 3D network and distribution: they are concentrated above all around the vessels, but also in the areas of looser connective tissue, characterized by wavy collagen fibers, large vessels and groups of adipocytes. Furthermore, in the dense connective areas of SF some vessels were noted: it is likely that they are branches of larger vessels located in loose connective areas. The nervous structures in the dense connective tissue of SF can be divided in two groups: thin nerve fiber bundles of 4.8 ± 2.6 μm diameter, and bigger nerve fascicles with a mean diameter of 21.1 ± 12.2 μm. These data are in accord with the values reported in the human hip joint area, in which the SF showed thin nervous structure of 19.1 ± 7.2 μm, with respect to large nerve bundles crossing the muscle with a diameter of 36.4 ± 13.4 μm (Fede et al., 2020). In this work, the only big nerves observed were located in the looser connective tissue areas, constituting passage nerve structures, compared to the small nerve fibers found in majority in the dense tissue. Unexpectedly, no Pacini corpuscles, which mediate vibration and pressure sensations, and Ruffini corpuscles, which perceive stretching, were found in these fasciae, also if they are usually described in the hypodermis (Cobo et al., 2021). The specific innervation of the superficial fascia and its strong relationship with the skin suggest that this innervation is part of the dermatomeric perception (Stecco et al., 2019).

In this study it was not possible to make a quantification and a comparison between the two investigated regions (abdomen and hip), for the low number of collected samples. However, the percentage of S-100 positive area calculated in the abdomen was 0.964% ± 0.110%, showing a bigger innervation with respect to the innervation of the hip area revealed by our group in a precedent article, equal to 0.22% ± 0.06% (Fede et al., 2020). The huge innervation by thin nerve fibers in the SF and the presence of single fibers in the middle of collagen bundles confirm‎ the sensory role of SF and its possible implications in nociception. These nervous structures were positive to S100, PGP 9.5, and TH.

토론

이 연구는

인간의 SF 신경 분포에 초점을 맞춘 최초의 조직학적 조사로,

고관절 부위의 SF가 피부 다음으로

가장 고도로 신경 분포된 조직임을 입증한 이전 연구

(피부>표층 근막>심부 근막>DAT>SAT; Fede et al., 2020)에 따라

자율 및 감각 신경 섬유가 모두 매우 많이 분포되어 있음을 보여줍니다.

일부 큰 신경 근막만이 표재성 근막을 통과하여 피부에 도달하고,

대부분의 신경 섬유는 혈관 주위에 위치하여

혈관 벽에 신경을 공급하거나 섬유 지방 조직에 매립되어 있습니다.

혈관 주위 신경 분포는 SF에서 신경총 구조를 나타냅니다. 플로팅 면역조직화학은 신경 섬유의 3D 네트워크와 분포를 강조하는 데 매우 유용했습니다. 신경 섬유는 무엇보다도 혈관 주변에 집중되어 있지만 물결 모양의 콜라겐 섬유, 큰 혈관 및 지방 세포 그룹이 특징인 느슨한 결합 조직 영역에도 집중되어 있습니다. 또한, SF의 조밀한 결합 영역에서 일부 혈관이 발견되었는데, 이는 느슨한 결합 영역에 위치한 더 큰 혈관의 가지일 가능성이 높습니다. SF의 치밀한 결합 조직의 신경 구조는 직경 4.8 ± 2.6 μm의 얇은 신경 섬유 다발과 평균 직경 21.1 ± 12.2 μm의 더 큰 신경 근막의 두 그룹으로 나눌 수 있습니다. 이러한 데이터는 인간의 고관절 부위에서 보고된 값과 일치하는데, SF는 19.1 ± 7.2 μm의 얇은 신경 구조를 보였고, 직경 36.4 ± 13.4 μm의 근육을 가로지르는 큰 신경 다발과 관련하여 (Fede et al., 2020)에서 보고된 값과 일치합니다. 이 연구에서 관찰된 유일한 큰 신경은 치밀한 조직에서 대부분 발견되는 작은 신경 섬유와 비교하여 통로 신경 구조를 구성하는 느슨한 결합 조직 영역에 위치했습니다. 진동과 압력 감각을 매개하는 파치니 소체와 스트레칭을 감지하는 루피니 소체는 일반적으로 피하에서 설명되는 경우에도 예기치 않게 이 근막에서 발견되지 않았습니다(Cobo et al., 2021). 표재성 근막의 특정 신경 분포와 피부와의 강한 관계는이 신경 분포가 피부 지각의 일부임을 시사합니다 (Stecco et al., 2019).

이 연구에서는 수집된 샘플 수가 적기 때문에 두 조사 부위(복부와 엉덩이)를 정량화하여 비교할 수 없었습니다. 그러나 복부에서 계산된 S-100 양성 면적의 비율은 0.964% ± 0.110%로, 선행 논문에서 우리 그룹이 밝힌 고관절 부위의 신경 분포(0.22% ± 0.06%)에 비해 더 큰 신경 분포가 나타났습니다(Fede et al., 2020). SF의 얇은 신경 섬유에 의한 거대한 신경 분포와 콜라겐 다발 중간에 단일 섬유의 존재는 SF의 감각 역할과 통각 수용에 대한 가능한 의미를 확인시켜줍니다. 이러한 신경 구조는 S100, PGP 9.5 및 TH에 양성 반응을 보였습니다.

The positivity to TH-staining confirmed the presence of a sympathetic autonomic nervous system inside the fascia, suggesting the possible role of the autonomic nervous system in the regulation of vascularization of the superficial fascia, but probably also of the more superficial layers, as the skin (Sheng and Zhu, 2018). The relative percentage of autonomic innervation in SF samples was equal to 33.82%, not so distant to the amount reported by Mense, who affirmed that approximately 40% of the entire fascia innervation consisted of postganglionic sympathetic fibers (Mense, 2019). Probably, the majority of these fibers are vasoconstrictors; however, some of the sympathetic endings seem to serve an unknown function as they do not terminate on the vessels, as demonstrated also in this study. This was reported also by a recent work by Neuhuber and Jänig, who demonstrated the presence of Aδ and C fibers and autonomic nervous fibers in thoracolumbar fascia of rats not associated with blood vessels, but inside the connective tissue with a probable trophic activity (Neuhuber and Jänig, 2017). A recent work (Larsson and Nagi, 2022) highlighted the role in the bidirectional pain modulation of some unmyelinated C-fibers, termed C low-threshold mechanoreceptors (C-LTMRs), positive for TH staining, both in human and animal studies, especially in the skin. A role of some TH-positive nervous fibers in the connective tissue of SF as pain modulator mechanoreceptors, which allow to mediate mechanical allodynia evoked by touch in conditions such as inflammatory pain, cannot be excluded. The sympathetic activity may have also a role in the control of fascial tone: Staubesand and Li (1996) proposed a close connection between fascial stiffness and sympathetic activation. By this way, the chronic stress can affect the basal tone of the fascial tissue, by the activation of the autonomic nervous system: recent works demonstrated, in fact, the influence of the sympathetic nervous system on the TGF-β1 expression‎, which in turn stimulates myofibroblast contractility (Bhowmick et al., 2009; Schleip et al., 2019). So, the presence of TH-positive fibers helps to state that a condition of stress, a trauma, or a sudden change in temperature may increase the sympathetic activity not only in the skin, but also in the superficial fascia (Scheff and Saloman, 2021; Ishikawa and Furuyashiki, 2022). This can help to understand how a state of chronic stress may create a change of the superficial fascia, causing some alterations in thermoregulation, lymphatic flow and venous circulation. The changes caused by the chronic stress can also influence the immune system: the TGF-β1/Smad2/3/Foxp3 axis was remarkably activated following chronic stress, causing lymphocyte apoptosis and immunosuppression (Zhang et al., 2018). These aspects can suggest a possible involvement of the SF in the mechanism of fibromyalgia: Evdokimov et al. (2020) demonstrated that in patients affected by fibromyalgia the dermal nerve fiber length of fibers with vessel contact was reduced, suggesting a possible relationship between sympathetic neurons and impaired thermal tolerance commonly reported by fibromyalgic patients.

Also the presence of a scar in the skin can influence an alteration of the SF: scars are in fact more severe and painful when the subcutaneous fascia beneath the dermis is injured upon surgical or traumatic wounding. A recent ultrasound imaging study revealed a thickening of the subcutaneous tissue of an injured knee in a young female patient (right/injured= 2.33 mm, left = 1.31 mm) and, in particular, of the SF with hyperechoic thickening of the retinacula cutis, causing significant pain (Pirri et al., 2020). It was demonstrated that the fascial tissue contains specialized sentry fibroblasts, which collectively migrate after injury, progressively causing a contraction of the skin and scar formation (Correa-Gallegos et al., 2019; Jiang et al., 2020). It cannot be excluded that the autonomic innervation may have a role in this process of regulation of scar formation.

Finally, the huge innervation of the SF can help to assume that a manual light superficial massage can have effects on the autonomic nervous system, helping to improve the symptoms related to dysfunction of the superficial venous system, or thermo-regulation. In conclusion, we can affirm that the SF has a clear and distinct anatomical entity and a specific innervation, which should be considered to improve grading and developing of different manual approach for treatments of fascial dysfunctions, and the understanding of how some factors like temperature or manual therapies can have an impact on nociception and sensitivity of the fascia.

Limitations and Further Research

Several limitations of this work should be declared. First, our analysis was based on only a small population sample: further studies with larger sample sizes from various anatomical regions are needed to establish the reliability and validity of our findings. Through a systematic study of the innervation of the SF, it will be possible to highlight some hypothetical variations due to the topographic region. Furthermore, it will be useful to broaden the eval‎uation of the innervation also to SAT and DAT in the same experimental conditions, to visualize possible variations with respect to the SF, such as the presence of Pacini and Ruffini corpuscles. It should also be added that by this work we did not use specific markers (anti-SP, CGRP or NGF), to demonstrate the presence of neuropeptides in the varicosities of the axons, so we have not demonstrated with certainty the presence of nerve terminals in the SF. Anyway, the small diameter of the nerve fibers inside the tissue and the aspects with varicosities, as indicated in the literature (Mense, 2019) and as visible by PGP9.5 staining (Figures 4C,F, 5C,F), lead to affirm that these are not passing fibers but a specific innervation of the superficial fascia. In the next future, a systematic and specific study will allow to identify the presence of nerve endings. Lastly, further studies are necessary to analyze the presence of nociceptive fibers in the SF, to better understand the role of this tissue in pain perception and consequently to better target physical therapies.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Ethics Statement

The studies involving human participants were reviewed and approved by Ethical Committee for clinical trials in the province of Padua. The patients/participants provided their written informed consent to participate in this study.

Author Contributions

CF: manuscript drafting, image analysis, and immunohistochemistry. LP: responsible for samples fixation, embedding, and for immunostainings. CP: participation in manuscript writing, image analysis, and statistical analyisis. WN and RS: participation in immunohistochemistry coordination and manuscript writing and editing. CT and CB: fascia collecting from the volunteers. RDC and CS: participation in manuscript writing and editing, and in work coordination. All authors contributed to the article and approved the submitted version.

Funding

This research work was supported by Verein zur Förderung der Faszienforschung e.V. (Fascia Research Charity Assoc.).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be eval‎uated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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