Evidence of a new hidden neural network into deep fasciae.

It is recognized that different fasciae have different type of innervation, but actually nothing is known about the specific innervation of the two types of deep fascia, aponeurotic and epymisial fascia. In this work the aponeurotic thoracolumbar fascia and the epymisial gluteal fascia of seven adult C57-BL mice were analysed by Transmission Electron Microscopy and floating immunohistochemistry with the aim to study the organization of nerve fibers, the presence of nerve corpuscles and the amount of autonomic innervation. The antibodies used were Anti-S100, Anti-Tyrosine Hydroxylase and Anti-PGP, specific for the Schwann cells forming myelin, the sympathetic nerve fibers, and the peripheral nerve fibers, respectively. The results showed that the fascial tissue is pervaded by a rhomboid and dense network of nerves. The innervation was statistically significantly lower in the gluteal fascia (2.78 ± 0.6% of positive area, 140.3 ± 31.6/mm2 branching points, nerves with 3.2 ± 0.6 mm length and 4.9 ± 0.2 µm thickness) with respect to the thoracolumbar fascia (9.01 ± 0.98% of innervated area, 500.9 ± 43.1 branching points/mm2, length of 87.1 ± 1.0 mm, thickness of 5.8 ± 0.2 µm). Both fasciae revealed the same density of autonomic nerve fibers (0.08%). Lastly, corpuscles were not found in thoracolumbar fascia. Based on these results, it is suggested that the two fasciae have different roles in proprioception and pain perception: the free nerve endings inside thoracolumbar fascia may function as proprioceptors, regulating the tensions coming from associated muscles and having a role in nonspecific low back pain, whereas the epymisial fasciae works to coordinate the actions of the various motor units of the underlying muscle.

Imaging of muscle activity-induced morphometric changes in fibril network of myofascia by two-photon microscopy.

Myofascia, deep fascia enveloping skeletal muscles, consists of abundant collagen and elastin fibres that play a key role in the transmission of muscular forces. However, understanding of biomechanical dynamics in myofascia remains very limited due to less quantitative and relevant approaches for in vivo examination. The purpose of this study was to evaluate the myofascial fibril structure by means of a quantitative approach using two-photon microscopy (TPM) imaging in combination with intravital staining of Evans blue dye (EBD), a far-red fluorescence dye, which potentially labels elastin. With focus on myofascia of the tibial anterior (TA) muscle, the fibril structure intravitally stained with EBD was observed at the depth level of collagen fibrous membrane above the muscle belly. The EBD-labelled fibril structure and orientation in myofascia indicated biomechanical responses to muscle activity and ageing. The orientation histograms of EBD-labelled fibrils were significantly modified depending upon the intensity of muscle activity and ageing. Moreover, the density of EBD-labelled fibrils in myofascia decreased with habitual exercise but increased with muscle immobilization or ageing. In particular, the diameter of EBD-labelled fibrils in aged mice was significantly higher. The orientation histograms of EBD-labelled fibrils after habitual exercise, muscle immobilization and ageing showed significant differences compared to control. Indeed, the histograms in bilateral TA myofascia of exercise mice made simple waveforms without multiple sharp peaks, whilst muscular immobilization or ageing significantly shifted a histogram with sustaining multiple sharp peaks. Therefore, the dynamics of fibre network with EBD fluorescence in response to the biomechanical environment possibly indicate functional tissue adaptation in myofascia. Furthermore, on the basis of the knowledge that neutrophil recruitment occurs locally in working muscles, we suggested the unique reconstruction mechanism involving neutrophilic elastase in the myofascial fibril structure. In addition to the elastolytic susceptibility of EBD-labelled fibrils, distinct immunoreactivities and activities of neutrophil elastase in the myofascia were observed after electric pulse stimulation-induced muscle contraction for 15 min. Our findings of EBD-labelled fibril dynamics in myofascia through quantitative approach using TPM imaging and intravital fluorescence labelling potentially brings new insights to examine muscle physiology and pathology.

Features of In Vitro Degradation and Physical Properties of a Biopolymer and In Vivo Tissue Reactions in Comparison with Polypropylene.

We compared in vitro degradation and physical properties of polypropylene and a biodegradable polymer synthesized by electrospinning and consisting of 65% polycaprolactone and 35% polytrimethylene carbonate as a possible alternative material for use in surgery for pelvic floor muscle failure. Samples of the studied polymers were implanted to 10 male Wistar rats into the interfascial space on the back (polypropylene on the right side and biodegradable polymer on the left side). The synthesized biopolymer was characterized by elongation and tear resistance, similar to those of polypropylene. During the period from the third to the sixth month after implantation, the area of fibrosis around individual polypropylene and biopolymer fibers increased by 16.7 and 107.9%, respectively, while remaining reduced compared to polypropylene. The total fibrosis area in 6 months after implantation of polypropylene and biopolymer samples significantly increased by 18% (p=0.0097) and 48% (p=0.05), respectively, i.e. fibrosing processes were more intense in case of biopolymer. Induction of more pronounced fibrosis can be an advantage of the synthesized biopolymer when choosing the material for fabrication of implants and their use for correction of incompetence of the ligamentous and muscular apparatus.

Morphological study of human facial fascia and subcutaneous tissue structure by region through SEM observation.

Fascia of the facial area is contiguous between fat tissues of the subcutaneous and connective tissue layers and does not envelope the muscle surface like other parts of the human body. This structure is called the superficial musculoaponeurotic system (SMAS), which is accepted as an international anatomical terminology. This special structure is commonly used to pull facial muscles during plastic surgeries such as a face lift. Most reports regarding the facial subcutaneous tissue structure including SMAS are in the field of plastic surgery, and only a few studies from a morphological perspective has been reported. Since the facial fascia does not envelope the muscular surface layer which is different from the deep fascia found on the general skeletal muscle surface, a clear definition of this structure has not been established yet. The purpose of this study was to clearly identify the basic morphological structure of the subcutaneous tissue layer containing the SMAS three-dimensionally through a scanning electron microscope using dissected specimen rather than living subjects. Moreover, this study explores structural differences among seven aging facial areas; thereby further clarifying the properties of the structure and add clinical significance and considerations.

Commentaries on the publication entitled: "Structure and distribution of an unrecognized interstitium in human tissues" by Benias et al. (2018)

The claim made in this publication of the existence of a hitherto unknown interstitial space is based on studies with sample-based confocal laser endo-microscopy (pCLM). Due to postings on various web portals (New Cellvizio, EurekAlert, Google Scholar,...) the alleged discovery has found great resonance. Nevertheless, there are several critical issues in this publication, the most important being that this is not the discovery of an "unrecognized" interstitium as it has, in fact, been known for a long time

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Reply to "Commentaries on the publication entitled: 'Structure and distribution of an unrecognized interstitium in human tissues' by Benias et al. (2018)

We appreciate the time and attention paid to our paper by Prof. Mestres-Ventura and similarly appreciate the opportunity to respond to his concerns. We would like to address what we believe are several fundamental misunderstandings in his commentary.1. Scale: The most significant misunderstanding is one of scale. The schematic (Fig. 1) provided by Prof. Mestres-Ventura is (per personal communication) at the nano scale, while in vivo microscopy of extrahepatic bile duct and dermis shows that the collagen bundles we report are at the micron scale, each containing many individual fibrils at the nanometer scale. Indeed, examining the tissues described in our paper - submucosae, dermis and subcutaneous fascia - fresh in resected specimens or intraoperatively, we find that the structures we describe are visible at the macroscopic level (if one leans in closely enough). In other words, they are macroscopic, not microscopic. Prof. Mestres-Ventura, in summarizing the prior pCLE work of Wallace and Fockens, which he notes is similar to ours, states "the 'holes' shown under intravital microscopy and in cryofixed samples are huge (over 20 µm)" This is exactly our point - we were surprised as well at the scale of these structures, as this has not been well appreciated in the past

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Evidence for novel tubular-bundle structures entangled in the fascia of the inner abdominal wall of a rat.

After incubation with Janus Green B in the peritoneal cavities of rats, lymph-vessel-like structures (LSs) were noticed under a stereomicroscope to run parallel to each other with rectangular branches on the inner abdominal wall; rarely were these LSs seen to connect with peritoneal LSs. These LSs were identified by using fluorescence microscopy (FM) at a magnification of 1,000X and confocal laser scanning microscopy (CLSM) to be lymph-vessel-like bundle structures (LBSs). Serial cross-sections of these LBSs were microscopically examined by using hematoxylin and eosin staining, Mattson trichrome staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry (IHC) with Lyve 1 and CD 31. The histology data from these LBSs revealed such novel characteristics as parallel clusters of live cells wrapped by collagen fibers of the fascia and an IHC different from those of lymphatic and blood vessels, being the novel bundle structures (NBSs). Under FM and CLSM with optical sections, some of the NBSs were observed to be able to swell like tiny balloons, implying that the bundle structures were hollow. Moreover, transmission electron microscopy images of two different cross-sections of an NBS showed it to be composed of four parallel tubules involving three kinds of sinuses with neither axons nor Schwann cells in the outermost wall, thus being a novel tubular-bundle structure (NTBS). The results of this research make evident with high repeatability (10/11) that beyond the orthodoxy of a single-tube system of blood and lymph vessels, a system of NTBSs is widely entangled in the fascia of the inner abdominal wall of a rat. Thus, the author suggests that NTBS-related functions and the entire NTBS network should be explored.

Structure and Distribution of an Unrecognized Interstitium in Human Tissues.

Confocal laser endomicroscopy (pCLE) provides real-time histologic imaging of human tissues at a depth of 60-70 µm during endoscopy. pCLE of the extrahepatic bile duct after fluorescein injection demonstrated a reticular pattern within fluorescein-filled sinuses that had no known anatomical correlate. Freezing biopsy tissue before fixation preserved the anatomy of this structure, demonstrating that it is part of the submucosa and a previously unappreciated fluid-filled interstitial space, draining to lymph nodes and supported by a complex network of thick collagen bundles. These bundles are intermittently lined on one side by fibroblast-like cells that stain with endothelial markers and vimentin, although there is a highly unusual and extensive unlined interface between the matrix proteins of the bundles and the surrounding fluid. We observed similar structures in numerous tissues that are subject to intermittent or rhythmic compression, including the submucosae of the entire gastrointestinal tract and urinary bladder, the dermis, the peri-bronchial and peri-arterial soft tissues, and fascia. These anatomic structures may be important in cancer metastasis, edema, fibrosis, and mechanical functioning of many or all tissues and organs. In sum, we describe the anatomy and histology of a previously unrecognized, though widespread, macroscopic, fluid-filled space within and between tissues, a novel expansion and specification of the concept of the human interstitium.

Mechanistic basis of manual therapy in myofascial injuries. Sonoelastographic evolution control.

The term myofascia is referred to the skeleton of muscle fibres organized as an interconnected 3D network that surrounds and connects the musculoskeletal system. Extracellular matrix muscle is relevant in tissue structural support and transmission of mechanical signals between fibres and tendons. Acute and chronic musculoskeletal injuries (muscle strain) are one of the major problems faced by those who practice any type of sport, regardless of whether they are professionals or amateurs. Therapeutic boarding is of uncertain value in most cases because there are many contributing factors such as type, severity, functional implication of the damaged tissue, progression or risk of relapse. Different studies suggest that the musculoskeletal cell matrix is essential for the development, maintenance and regeneration of skeletal muscle. In this article, we highlight the action of "non-contractile" structures, in particular the myofascial system or muscle fascia, which can be responsible for the pathophysiology and healing process of muscular injuries. Manual therapy plays a predominant role in the treatment of these types of injuries and is key in the process of obtaining a scar capable of transmitting mechanical information. The scientific basis of this process is described in this article. Through real-time sonoelastography we have accurate information regarding the current stage of the repair process and, thus, guide our treatment at all times. Some new concepts are introduced, including local elasticity, the relationship between fascial pretension and the different stages of the physiological myofascia repair process, scar modelling technique, and sonoelastographic evolution control.

The muscular force transmission system: role of the intramuscular connective tissue.

The objective of this review is to analyze in detail the microscopic structure and relations among muscular fibers, endomysium, perimysium, epimysium and deep fasciae. In particular, the multilayer organization and the collagen fiber orientation of these elements are reported. The endomysium, perimysium, epimysium and deep fasciae have not just a role of containment, limiting the expansion of the muscle with the disposition in concentric layers of the collagen tissue, but are fundamental elements for the transmission of muscular force, each one with a specific role. From this review it appears that the muscular fibers should not be studied as isolated elements, but as a complex inseparable from their fibrous components. The force expressed by a muscle depends not only on its anatomical structure, but also the angle at which its fibers are attached to the intramuscular connective tissue and the relation with the epimysium and deep fasciae.

Electron-microscopic imaging of endothoracic fascia in the thoracic paravertebral space in rats.

BACKGROUND AND OBJECTIVES: Anesthesia and analgesia with paravertebral block are reportedly variable. Existence of an endothoracic fascia has been proposed as one of the possible mechanisms leading to variability. We undertook an electron-microscopy imaging study to investigate the endothoracic fascia in the thoracic paravertebral space (TPS) in rats. METHODS: Male Wistar rats were studied in accordance with the principles of laboratory animal care. After the rats were euthanized in a CO2 chamber, the thoracic paravertebral tissues were removed en bloc and cut into consecutive transverse sections of approximately 3 mm. Stereomicroscopy and electron-microscopy assessments were performed by 2 independent observers. RESULTS: The endothoracic fascia was consistently identified in all specimens. The fascia was located between the parietal pleura and the innermost intercostal muscles or ribs. Its thickness ranged from 15 to 27 µm (mean, 20 ± 3 µm). The endothoracic fascia divided the TPS in 2 compartments: one, extrapleural and anterolateral (EPC); another, subendothoracic and posteromedial (SETC). The spinal nerves with their ganglia were found within SETC, whereas the sympathetic ganglia were consistently located within the EPC. CONCLUSIONS: The endothoracic fascia in rats appears to divide the TPS into EPC and SETC. These anatomic characteristics may have implications in thoracic paravertebral blockade.

Apoptosis-like cell death induction and aberrant fibroblast properties in human incisional hernia fascia.

Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo.

Management of Dupuytren contracture with ultrasound-guided lidocaine injection and needle aponeurotomy coupled with osteopathic manipulative treatment.

Dupuytren contracture is a debilitating disease that characteristically presents as a firm nodularity on the palmar surface of the hand with coalescing cords of soft tissue on the webs and digits. With few nonsurgical modalities providing clinical benefits, open surgical procedures are the standard of care for patients with this condition. However, recent studies have associated surgical intervention with many complications, necessitating further exploration of nonsurgical treatment options. We describe the case of a 64-year-old woman who presented with decreased extension of the fourth and fifth digits on the upper extremities bilaterally; previous conservative treatment regimens had been unsuccessful. After a diagnostic ultrasound, the patient was diagnosed as having Dupuytren contracture and underwent 5 treatments consisting of ultrasound-guided dry-needle aponeurotomy, lidocaine injections, and osteopathic manipulative treatment. During the fifth treatment session, the patient experienced dramatic relief of her symptoms after a palpable release during the manual manipulation portion of her therapeutic regimen. At 2-week follow-up, the patient was symptom-free. Based on this desirable outcome, the authors suggest future research be directed at minimally invasive therapeutic options in the management of Dupuytren contracture.

The microvacuolar system: how connective tissue sliding works.

The term 'fascia' has been applied to a large number of very different tissues within the hand. These range from aligned ligamentous formations such as the longitudinal bands of the palmar fascia or Grayson's and Cleland's ligaments, to the loose packing tissues that surround all of the moving structures within the hand. In other parts of the body the terms 'superficial' and 'deep fascia' are often used but these have little application in the hand and fingers. Fascia can be divided into tissues that restrain motion, act as anchors for the skin, or provide lubrication and gliding. Whereas the deep fascia is preserved and easily characterized in anatomical dissection, the remaining fascial tissue is poorly described. Understanding its structure and dynamic anatomy may help improve outcomes after hand injury and disease. This review describes the sliding tissue of the hand or the 'microvacuolar system' and demonstrates how movement of tissues can occur with minimal distortion of the overlying skin while maintaining tissue continuity.

High second harmonic generation signal from muscles and fascia pig's muscles using the two-photon laser scanning microscope.

I have provided update to our two photon laser scanning microscope by adding new technique which enables us to simultaneously measured the second harmonic generation signals in the forward and backward directions; in the meantime, one can measure the two photon excitations fluorescence if the materials produce fluorescence. In the present work, the fascia muscles, muscles of pig and pig's skin were used. I found that these materials produced high second harmonic generation signal in both directions. These measurements show that the second harmonic generation strongly depends on the state of the polarization of the laser light and the orientation of the dipole moment in the molecules that interact with the laser light. It is therefore advantageous to control the laser's state of polarization, to maximize second harmonic generation. The novelty of this work is to establish new multi-functional technique by combing three platforms of laser scanning microscopy - the fluorescence microscopy, harmonic generation microscopy and polarizing microscopy in which one can use the second harmonic imaging to investigate the true architecture of the sensitive samples and the samples which do not produce auto-fluorescence. Moreover investigation of the new sample needs to look at all details of the true architecture of the sample. Thereby the sample will be exposed to the laser radiation more than the well-known sample, and that will cause photo-bleaching and photo-damage. Since the second harmonic generation does not undergo from photo-bleaching and photo-damage it will be the promising technique for investigating the sensitive and new samples. Then one can move to acquire fluorescence images after good investigation of the true architecture of the sample by the SH imaging.

The role of skin and subcutaneous tissues in Dupuytren's contracture: an electron microscopic observation.

OBJECTIVE: To determine the relationship between the overlying tissues and recurrence of Dupuytren's contracture. METHODS: Forty-three patients (68 hands) who accepted surgical treatment were divided into two groups according to treatment methods: partial fasciectomy or dermofasciectomy and full-thickness skin graft. Diseased palmar fascia, subcutaneous tissues and skin obtained during surgery were then assessed by electron microscopy. RESULTS: All patients were followed up. None of the hands which accepted dermofasciectomy and full-thickness skin grafting recurred, while 46.4% of the hands which accepted partial fasciectomy recurred. Under electron microscopic observation, myofibroblasts were found in the skin and subcutaneous tissues. CONCLUSION: The overlying tissues play an important role in Dupuytren's contracture, which may be a reason for recurrence of this condition after surgical treatment.

Multimodal nonlinear optical imaging of collagen arrays.

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.

Different crimp patterns in collagen fibrils relate to the subfibrillar arrangement.

Collagen fibril ultrastructure and course were examined in different connective tissues by PLM, SEM, TEM, and AFM. In tendons, collagen fibrils were large and heterogeneous with a straight subfibrillar arrangement. They ran densely packed, parallel, and straight changing their direction only in periodic crimps where fibrils showed a local deformation (fibrillar crimps). Other tissues such as aponeurosis, fascia communis, skin, aortic wall, and tendon and nerve sheaths showed thinner uniform fibrils with a helical subfibrillar arrangement. These fibrils appeared in parallel or helical arrangement following a wavy, undulating course. Ligaments showed large fibrils as in tendon, with fibrillar crimps but less packed. Thinner uniform-sized fibrils also were observed. Fibrillar crimps seem to be related to the subfibrillar arrangement being present only in large fibrils with a straight subfibrillar arrangement. These stiffer fibrils respond mainly to unidirectional tensional forces, whereas the flexible thinner fibrils with helical subfibrils can accommodate extreme curvatures without harm, thus responding to multidirectional loadings.

The role of backscattering in SHG tissue imaging.

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.

The deep fascia in response to leg lengthening with particular reference to the tension-stress principle.

OBJECTIVE: To investigate the morphological changes of deep fascia subjected to distraction in a rabbit model of leg lengthening. METHODS: The animal model of leg lengthening was established in 20 New Zealand white rabbits using a unilateral external fixator with 4 half pins medially fixed to the tibia, and osteotomy was performed between the second and the third pins. The distraction was initiated 7 days after the osteotomy procedure, with the rate of 1 and 2 mm/d in 2 steps, and proceeded until 10% and 20% increases in the initial length of the tibia had been achieved. The deep fascia samples were studied by the hematoxylin-eosin stain, the Masson-trichome staining, and the JEM2000EX electron microscopy. RESULTS: Under light, the cross section of normal deep fascia without distraction consisted of 3 layers. Whereas in the longitudinal sections, the deep fascia consisted of wavy collagen fibers. The normal deep fascia consisted of fibrocyte and collagenous fibrils under electron microscopy. After leg lengthening, the morphology of the fascia distracted at each rate changed. Under light, the fascia distracted at each rate kept the normal 3 layers in the cross sections. The fascia subjected to distraction at a rate of 2 mm/d showed injuries of collagen fibers. Under electron microscopy, the fascia distracted at a rate of 2 mm/d showed active metabolism to repair the necrotic collagenous fibrils. Whereas the fascia subjected to distraction at a rate of 1 mm/d showed regenerative changes. The endotheliocyte of capillaries within the deep fascia subjected to distraction at a rate of 1mm/d with 20% lengthening of tibia was metabolically active. CONCLUSIONS: The tension stress, which is applied in leg lengthening and deformity correction as described by Ilizarov, has a great effect upon the fascia, which is always related to the function of the involved limb. The appropriate regimen of distraction at the rate of 1 mm/d with 20% lengthening of tibia leads to the regenerative changes in deep fascia, ultimately close to the morphological structure of normal under the condition of this investigation.