The suprapatellar fat pad: A histotopographic comparative study.

The suprapatellar fat pad is an adipose tissue located in the anterior knee whose role in osteoarthritis is still debated. Considering that anatomy drives function, the aim of this histotopographic study was to investigate the specific morphological features of the suprapatellar fat pad versus the infrapatellar fat pad in the absence of osteoarthritis, for a broad comparative analysis. Suprapatellar fat pad and infrapatellar fat pad tissue samples (n = 10/group) underwent microscopical/immunohistochemical staining and transmission electron microscopy analysis; thus, tissue-specific characteristics (i.e., vessels and nerve endings presence, lobuli, adipocytes features, septa), including extracellular matrix proteins prevalence (collagens, elastic fibers), were focused. Multiphoton microscopy was also adopted to evaluate collagen fiber orientation within the samples by Fast Fourier Transform (coherency calculation). The absence of inflammation was confirmed, and comparable counted vessels and nerve endings were shown. Like the infrapatellar fat pad, the suprapatellar fat pad appeared as a white adipose tissue with lobuli and septa of comparable diameter and thickness, respectively. Tissue main characteristics were also proved by both semithin sections and transmission electron microscopy analysis. The suprapatellar fat pad adipocytes were roundish and with a smaller area, perimeter, and major axis than that of the infrapatellar fat pad. The collagen fibers surrounding them showed no significant difference in collagen type I and significantly higher values for collagen type III in the infrapatellar fat pad group. Regarding the septa, elastic fiber content was statistically comparable between the two groups, even though more represented by the suprapatellar fat pad. Total collagen was significantly higher in the infrapatellar fat pad and comparing collagen type I and type III they were similarly represented in the whole cohort despite collagen type I appearing to be higher in the infrapatellar fat pad than in the suprapatellar fat pad and vice versa for collagen type III. Second harmonic generation microscopy confirmed through coherency calculation an anisotropic distribution of septa collagen fibers. From a mechanical point of view, the different morphological characteristics determined a major stiffness for the infrapatellar fat pad with respect to the suprapatellar fat pad. This study provides, for the first time, a topographic description of the suprapatellar fat pad compared to the infrapatellar fat pad; differences between the two groups may be attributed to a different anatomical location within the knee; the results gathered here may be useful for a more complete interpretation of osteoarthritis disease, involving not only cartilage but the whole joint.

Myofascial junction: Emerging insights into the connection between deep/muscular fascia and muscle.

Muscles and fasciae are mutually connected and are influenced by force transmission. However, the anatomical connectivity and histological features of these structures remain unclear. The aim of this study was to assess the evidence for connection between muscles and deep/muscular fasciae. We assessed this relationship in different topographical regions of human cadavers and in mice. The results showed that myofascial junctions (MFJ) were made up of collagen I immune-positive structures occupying an average area of 5.11 ± 0.81 µm2, distributed in discrete regions at the interface between muscle and fascia with an average density of 9.7 ± 2.51 MFJ/mm and an average inclination angle of 35.25 ± 1.52°. These specialized structures also showed collagen III and HA immunopositivity and the presence of elastic fibers. The human myofascial junction can be visualized, opening emerging insights into the connection between deep/muscular fascia and muscle.

Duodenal alpha-Synuclein Pathology and Enteric Gliosis in Advanced Parkinson's Disease.

BACKGROUND: The role of the gut-brain axis has been recently highlighted as a major contributor to Parkinson's disease (PD) physiopathology, with numerous studies investigating bidirectional transmission of pathological protein aggregates, such as α-synuclein (αSyn). However, the extent and the characteristics of pathology in the enteric nervous system have not been fully investigated. OBJECTIVE: We characterized αSyn alterations and glial responses in duodenum biopsies of patients with PD by employing topography-specific sampling and conformation-specific αSyn antibodies. METHODS: We examined 18 patients with advanced PD who underwent Duodopa percutaneous endoscopic gastrostomy and jejunal tube procedure, 4 untreated patients with early PD (disease duration <5 years), and 18 age- and -sex-matched healthy control subjects undergoing routine diagnostic endoscopy. A mean of four duodenal wall biopsies were sampled from each patient. Immunohistochemistry was performed for anti-aggregated αSyn (5G4) and glial fibrillary acidic protein antibodies. Morphometrical semiquantitative analysis was performed to characterize αSyn-5G4+ and glial fibrillary acidic protein-positive density and size. RESULTS: Immunoreactivity for aggregated α-Syn was identified in all patients with PD (early and advanced) compared with controls. αSyn-5G4+ colocalized with neuronal marker ß-III-tubulin. Evaluation of enteric glial cells demonstrated an increased size and density when compared with controls, suggesting reactive gliosis. CONCLUSIONS: We found evidence of synuclein pathology and gliosis in the duodenum of patients with PD, including early de novo cases. Future studies are required to evaluate how early in the disease process duodenal pathology occurs and its possible contribution to levodopa effect in chronic patients. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Hearing and Seeing Nerve/Tendon Snapping: A Systematic Review on Dynamic Ultrasound Examination.

Nerve/tendon snapping can occur due to their sudden displacement during the movement of an adjacent joint, and the clinical condition can really be painful. It can actually be challenging to determine the specific anatomic structure causing the snapping in various body regions. In this sense, ultrasound examination, with all its advantages (especially providing dynamic imaging), appears to be quite promising. To date, there are no comprehensive reviews reporting on the use of dynamic ultrasound examination in the diagnosis of nerve/tendon snapping. Accordingly, this article aims to provide a substantial discussion as to how US examination would contribute to 'seeing' and 'hearing' these pathologies' different maneuvers/movements.

Reference centers for tissue and body donations: Compulsory requirements in Italy.

Recent Italian legislation (Law No. 10/2020) establishes the legal, ethical and technical requirements governing how individuals can donate their bodies or tissues post-mortem for study, training, and scientific research purposes. A ministerial working group has recently approved some of the law's implementing rules relating to technical issues. The rules for implementing the new legislation, retrieved from the legal databases and translated into English, are discussed. For the first time in Italy, the law establishes compulsory requirements and minimum characteristics-in terms of logistics, safety, availability of staff, space, and equipment-for institutions to be recognized as reference centers for the conservation and use of bodies and tissues donated post-mortem for study, training and scientific research purposes. This makes it possible to standardize the features of such reference centers nationwide, and to coordinate their activities and potential future development, while ensuring basic operational efficiency and workplace safety, the provision of adequate facilities, buildings and equipment, and staff training. The law and its implementing rules discussed here are the first to establish criteria and compulsory requirements for centers receiving body and tissue donations in Italy. This is a step forward for the whole anatomy community, providing practical guidelines for body donation programs and related facilities in other countries too.

What's old is new again: The anatomical studies of Franklin P. Mall and the fascial-interstitial spaces.

Franklin Mall was one of the foremost scientists of the turn of the 19th century, an exemplary mentor as well as researcher, and his revolutionary contributions are still relevant today. Mall's early training in Leipzig with Wilhelm His and Carl Ludwig provided him with an unusual perspective on the integration of anatomy and physiology, and his interest in the links between structure and function guided the work he carried out after joining the faculty of the new Johns Hopkins University School of Medicine. Mall carried out innovative studies on the one hand using dye injection to trace blood and lymphatic supplies to different organs and on the other hand using "putrefaction" to digest tissues and study the organization of the reticular space, demonstrating that it was the underlying source of support for all the organs. These two studies of Mall's, carried out independently, provide the basis for modern studies integrating the understanding of fascia and interstitial spaces.

Blood supply to the superficial fascia of the abdomen: An anatomical study.

The aim of this study was to examine data demonstrating that Scarpa's fascia, a superficial fascia of the anterior abdominal wall, is a vascularized tissue. Specimens of the fascia of seven volunteers undergoing abdominoplasty surgical procedures at the Plastic Surgery Unit of the University of Padova Medical Center were collected. Fractal analysis and quantitative assessment of the vascular network of the fascia was carried out, exploiting the presence of blood in the vessels. Each sample was divided and processed for histological/immunohistochemical analysis (into 5 micron-paraffin embedded sections and cryo-sectioned free-floating samples) as well as for electron microscopy study. A rich vascular pattern forming a fine, dense meshwork with an area percentage of 6.20% ± 2.10% von Willebrand factor stained vessels was noted in all the specimens of the fascia examined; the area percentage of the αSMA-stained vessels was 2.93% ± 1.80%. The diameters of the vessels fell between the 13 and 65 µm range; the network was composed of arteries, veins, capillaries and lymphatic segments. Topological results showed that the vascular network within Scarpa's fascia is well branched (segments: 6615 ± 3070 and 8.40 ± 3.40 per mm2 ; crossing points: 3092 ± 1490 and 3.40 ± 1.90 per mm2 ). Fractal analysis (fractal dimension = 1.063 ± 0.10; lacunarity = 0.60 ± 0.10) revealed that this particular vascular network has an optimal spatial distribution and homogeneity occupying the entire space of the superficial fascia. These findings could undoubtedly be useful to plastic surgeons as well as to pain management specialists.

Connections between postparotid terminal branches of the facial nerve: An immunohistochemistry study.

It has been assumed that connections between the postparotid terminal branches of the facial nerve are purely motor. However, the nature of their fibers remains unexplored. The aim of this study is to determine whether these connections comprise motor fibers exclusively. In total 17 connections between terminal facial nerve branches were obtained from 13 different facial nerves. Choline acetyltransferase antibody (ChAT) was used to stain the fibers in the connections and determine whether or not all of them were motor. All connections contained ChAT positive and negative fibers. The average number of fibers overall was 287 (84-587) and the average proportion of positive fibers was 63% (37.7%-91.5%). In 29% of the nerves, >75% of the fibers were ChAT+ (strongly positive); in 52.94%, 50%-75% were ChAT+ (intermediately positive); and in 17.65%, <50% were ChAT+ (weakly positive). Fibers traveling inside the postparotid terminal cranial nerve VII branch connections are not exclusively motor.

From Muscle to the Myofascial Unit: Current Evidence and Future Perspectives.

The "motor unit" or the "muscle" has long been considered the quantal element in the control of movement. However, in recent years new research has proved the strong interaction between muscle fibers and intramuscular connective tissue, and between muscles and fasciae, suggesting that the muscles can no longer be considered the only elements that organize movement. In addition, innervation and vascularization of muscle is strongly connected with intramuscular connective tissue. This awareness induced Luigi Stecco, in 2002, to create a new term, the "myofascial unit", to describe the bilateral dependent relationship, both anatomical and functional, that occurs between fascia, muscle and accessory elements. The aim of this narrative review is to understand the scientific support for this new term, and whether it is actually correct to consider the myofascial unit the physiological basic element for peripheral motor control.

Detection of Mast Cells in Human Superficial Fascia.

The recent findings showed that the superficial fascia is a fibrous layer in the middle of hypodermis, richly innervated and vascularized, and more complex than so far demonstrated. This study showed the presence of mast cells in the superficial fascia of the human abdomen wall of three adult volunteer patients (mean age 42 ± 4 years; 2 females, 1 male), by Toluidine Blue and Safranin-O stains and Transmission Electron Microscopy. Mast cells are distributed among the collagen bundles and the elastic fibers, near the vessels and close to the nerves supplying the tissue, with an average density of 20.4 ± 9.4/mm2. The demonstration of the presence of mast cells in the human superficial fascia highlights the possible involvement of the tissue in the inflammatory process, and in tissue healing and regeneration processes. A clear knowledge of this anatomical structure of the hypodermis is fundamental for a good comprehension of some fascial dysfunctions and for a better-targeted clinical practice.

Bridging Gaps in Peripheral Nerves: From Current Strategies to Future Perspectives in Conduit Design.

In peripheral nerve injuries (PNI) with substance loss, where tensionless end-to-end suture is not achievable, the positioning of a graft is required. Available options include autografts (e.g., sural nerve, medial and lateral antebrachial cutaneous nerves, superficial branch of the radial nerve), allografts (Avance®; human origin), and hollow nerve conduits. There are eleven commercial hollow conduits approved for clinical, and they consist of devices made of a non-biodegradable synthetic polymer (polyvinyl alcohol), biodegradable synthetic polymers (poly(DL-lactide-ε-caprolactone); polyglycolic acid), and biodegradable natural polymers (collagen type I with/without glycosaminoglycan; chitosan; porcine small intestinal submucosa); different resorption times are available for resorbable guides, ranging from three months to four years. Unfortunately, anatomical/functional nerve regeneration requirements are not satisfied by any of the possible alternatives; to date, focusing on wall and/or inner lumen organization/functionalization seems to be the most promising strategy for next-generation device fabrication. Porous or grooved walls as well as multichannel lumens and luminal fillers are the most intriguing options, eventually also including the addition of cells (Schwann cells, bone marrow-derived, and adipose tissue derived stem cells) to support nerve regeneration. This review aims to describe common alternatives for severe PNI recovery with a highlight of future conduits.

A New Player in the Mechanobiology of Deep Fascia: Yes-Associated Protein (YAP).

Recent studies have demonstrated that fascial fibroblasts are susceptible to mechanical stimuli, leading to the remodeling of the extracellular matrix (ECM). Moreover, the extensive literature on Yes-associated protein (YAP) has shown its role in cell mechanics, linking cell properties, such as shape, adhesion, and size, to the expression of specific genes. The aim of this study was to investigate the presence of YAP in deep fascia and its activation after a mechanical stimulus was induced via a focal extracorporeal shockwave (fESW) treatment. Thoracolumbar fascia (TLF) samples were collected from eight patients (age: 30-70 years; four males and four females) who had undergone spine elective surgical procedures at the Orthopedic Clinic of University of Padova. YAP was measured in both tissue and TLF-derived fibroblasts through immunoblotting. COL1A1 and HABP2 gene expression were also evaluated in fibroblasts 2, 24, and 48 h after the fESW treatment. YAP was expressed in all the examined tissues. The ratio between the active/inactive forms (YAP/p-YAP) of the protein significantly increased in fascial fibroblasts after mechanical stimulation compared to untreated cells (p = 0.0022). Furthermore, COL1A1 and HABP2 gene expression levels were increased upon treatment. These findings demonstrate that YAP is expressed in the deep fascia of the thoracolumbar region, suggesting its involvement in fascial mechanotransduction processes, remodeling, regeneration, and fibrogenesis. This study indicates, for the first time, that YAP is a "new player" in the mechanobiology of deep fascia.

The transversoclasiotome: a novel instrument for examining the vertebral artery.

Opening the foramen transversarium of the cervical vertebrae is necessary for accessing the vertebral vessels. There are no specialist tools for cutting the anterior lamina of the transverse processes, and alternatives lead to questionable results. A novel tool, the transversoclasiotome, is described and tested. The literature and patent databases were systematically reviewed. A blueprint of the transversoclasiotome was created, and the prototype was tested through autopsy on ten fresh-frozen cadavers within our Body Donation Program. The transversoclasiotome consists of two delicate branches mounted as a scissor, one a cutting jaw and the other a knocker with a rounded tip, both angled 30° to the principal axis. The jaws shut, facing each other in parallel. The cutting jaw corresponds to a slit on the knocker profile without protruding beyond it even when entirely closed. It acts by cutting and wedging. The testing autopsies demonstrated its suitability for its purpose, with an adequate response to the pressure exerted on the bone lamina. The section cut cleanly, without sliding off while closing on the bone. The vertebral vessels were not injured either during instrument insertion or cutting. Their morphological features are described. The transversoclasiotome has been proven appropriate for sectioning the anterior lamina of transverse processes of the cervical vertebrae. It meets the needs of clinical anatomy in teaching and training clinicians or surgeons, forensic clinical anatomy during medico-legal investigation, and research.

Elastic Fibres in the subcutaneous tissue: Is there a difference between superficial and muscular fascia? A cadaver study.

BACKGROUND: In last years the role of fascia in proprioception and pain has been confirmed in numerous papers, but the real structure of fasciae is not still entirely known. To date, many studies have evaluated the elastic fibres in arteries, ligaments, lungs, epidermis and dermis, but only two studies exist about the elastic fibres in the fasciae, and they did not distinguish between superficial (in the subcutaneous tissue) and deep/muscular fasciae. The aim of the study was to assess the percentage of elastic fibres between superficial and deep fascia. MATERIALS AND METHODS: Three full thickness specimens (proximal, middle and distal respectively) were taken from each of four regions of the thigh of three non-embalmed cadavers: the anterior (Ant), the lateral (Lat), the posterior (Post) and the medial (Med) aspect. Thus, a total of 12 specimens were collected from each analysed thigh and histological Weigert Van Gieson stains was performed. Three sections per specimen were considered for the morphometric analysis. RESULTS: In all the specimens the superficial and deep fasciae were clearly recognizable. The difference in percentage of elastic fibres between superficial and deep fasciae in same region for all four was highly significant (p < 0.001). They are abundant in the superficial fascia than deep fascia. CONCLUSIONS: In the light of these findings is evident that the superficial (in the subcutaneous tissue) and deep fasciae have different elasticity. This difference may improve grading of fascial dysfunction in dermatological diseases as burns, scars and lymphedema to better plan treatments.

Fascial Innervation: A Systematic Review of the Literature.

Currently, myofascial pain has become one of the main problems in healthcare systems. Research into its causes and the structures related to it may help to improve its management. Until some years ago, all the studies were focused on muscle alterations, as trigger points, but recently, fasciae are starting to be considered a new, possible source of pain. This systematic review has been conducted for the purpose of analyze the current evidence of the muscular/deep fasciae innervation from a histological and/or immunohistochemical point of view. A literature search published between 2000 and 2021 was made in PubMed and Google Scholar. Search terms included a combination of fascia, innervation, immunohistochemical, and different immunohistochemical markers. Of the 23 total studies included in the review, five studies were performed in rats, four in mice, two in horses, ten in humans, and two in both humans and rats. There were a great variety of immunohistochemical markers used to detect the innervation of the fasciae; the most used were Protein Gene Marker 9.5 (used in twelve studies), Calcitonin Gene-Related Peptide (ten studies), S100 (ten studies), substance P (seven studies), and tyrosine hydroxylase (six studies). Various areas have been studied, with the thoracolumbar fascia being the most observed. Besides, the papers highlighted diversity in the density and type of innervation in the various fasciae, going from free nerve endings to Pacini and Ruffini corpuscles. Finally, it has been observed that the innervation is increased in the pathological fasciae. From this review, it is evident that fasciae are well innerved, their innervation have a particular distribution and precise localization and is composed especially by proprioceptors and nociceptors, the latter being more numerous in pathological situations. This could contribute to a better comprehension and management of pain.

The Effects of Aging on the Intramuscular Connective Tissue.

The intramuscular connective tissue plays a critical role in maintaining the structural integrity of the muscle and in providing mechanical support. The current study investigates age-related changes that may contribute to passive stiffness and functional impairment of skeletal muscles. Variations in the extracellular matrix in human quadriceps femoris muscles in 10 young men, 12 elderly males and 16 elderly females, and in the hindlimb muscles of 6 week old, 8 month old and 2 year old C57BL/6J male mice, were evaluated. Picrosirius red, Alcian blue and Weigert Van Gieson stainings were performed to evaluate collagen, glycosamynoglycans and elastic fibers. Immunohistochemistry analyses were carried out to assess collagen I, collagen III and hyaluronan. The percentage area of collagen was significantly higher with aging (p < 0.01 in humans, p < 0.001 in mice), mainly due to an increase in collagen I, with no differences in collagen III (p > 0.05). The percentage area of elastic fibers in the perimysium was significantly lower (p < 0.01) in elderly men, together with a significant decrease in hyaluronan content both in humans and in mice. No significant differences were detected according to gender. The accumulation of collagen I and the lower levels of hyaluronan and elastic fibers with aging could cause a stiffening of the muscles and a reduction of their adaptability.

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A.

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

Development of Two-Layer Hybrid Scaffolds Based on Oxidized Polyvinyl Alcohol and Bioactivated Chitosan Sponges for Tissue Engineering Purposes.

Oxidized polyvinyl alcohol (OxPVA) is a new polymer for the fabrication of nerve conduits (NCs). Looking for OxPVA device optimization and coupling it with a natural sheath may boost bioactivity. Thus, OxPVA/chitosan sponges (ChS) as hybrid scaffolds were investigated to predict in the vivo behaviour of two-layered NCs. To encourage interaction with cells, ChS were functionalized with the self-assembling-peptide (SAP) EAK, without/with the laminin-derived sequences -IKVAV/-YIGSR. Thus, ChS and the hybrid scaffolds were characterized for mechanical properties, ultrastructure (Scanning Electron Microscopy, SEM), bioactivity, and biocompatibility. Regarding mechanical analysis, the peptide-free ChS showed the highest values of compressive modulus and maximum stress. However, among +EAK groups, ChS+EAK showed a significantly higher maximum stress than that found for ChS+EAK-IKVAV and ChS+EAK-YIGSR. Considering ultrastructure, microporous interconnections were tighter in both the OxPVA/ChS and +EAK groups than in the others; all the scaffolds induced SH-SY5Y cells' adhesion/proliferation, with significant differences from day 7 and a higher total cell number for OxPVA/ChS+EAK scaffolds, in accordance with SEM. The scaffolds elicited only a slight inflammation after 14 days of subcutaneous implantation in Balb/c mice, proving biocompatibility. ChS porosity, EAK 3D features and neuro-friendly attitude (shared with IKVAV/YIGSR motifs) may confer to OxPVA certain bioactivity, laying the basis for future appealing NCs.

Intratracheal administration of mesenchymal stem cell-derived extracellular vesicles reduces lung injuries in a chronic rat model of bronchopulmonary dysplasia.

Early therapeutic effect of intratracheally (IT)-administered extracellular vesicles secreted by mesenchymal stem cells (MSC-EVs) has been demonstrated in a rat model of bronchopulmonary dysplasia (BPD) involving hyperoxia exposure in the first 2 postnatal weeks. The aim of this study was to evaluate the protective effects of IT-administered MSC-EVs in the long term. EVs were produced from MSCs following GMP standards. At birth, rats were distributed in three groups: (a) animals raised in ambient air for 6 weeks (n = 10); and animals exposed to 60% hyperoxia for 2 weeks and to room air for additional 4 weeks and treated with (b) IT-administered saline solution (n = 10), or (c) MSC-EVs (n = 10) on postnatal days 3, 7, 10, and 21. Hyperoxia exposure produced significant decreases in total number of alveoli, total surface area of alveolar air spaces, and proliferation index, together with increases in mean alveolar volume, mean linear intercept and fibrosis percentage; all these morphometric changes were prevented by MSC-EVs treatment. The medial thickness index for <100 µm vessels was higher for hyperoxia-exposed/sham-treated than for normoxia-exposed rats; MSC-EV treatment significantly reduced this index. There were no significant differences in interstitial/alveolar and perivascular F4/8-positive and CD86-positive macrophages. Conversely, hyperoxia exposure reduced CD163-positive macrophages both in interstitial/alveolar and perivascular populations and MSC-EV prevented these hyperoxia-induced reductions. These findings further support that IT-administered EVs could be an effective approach to prevent/treat BPD, ameliorating the impaired alveolarization and pulmonary artery remodeling also in a long-term model. M2 macrophage polarization could play a role through anti-inflammatory and proliferative mechanisms.

An anatomical comparison of the fasciae of the thigh: A macroscopic, microscopic and ultrasound imaging study.

Although the number of Ultrasound (US) imaging studies investigating the fascial layers are becoming more numerous, the majority tend to use different reference points and terminology to describe their findings. The current work set out to compare macroscopic and microscopic data of specimens of the fascial layers of the thigh with US imaging findings. Specimens of the different fascial layers of various regions of the thigh were collected for macroscopic and histological analyses from three fresh cadavers and compared with in vivo US images of the thighs of 20 healthy volunteers. The specimens showed that the subcutaneous tissue of the thigh is made up of three layers: a superficial adipose layer, a membranous layer/superficial fascia, and a deep adipose layer. The deep fascia is composed of an aponeurotic fascia, which envelops all the thigh muscles and is laterally reinforced by the iliotibial tract and an epimysial fascia, which is specific for each muscle. The morphometric measurements of the thickness of the superficial fascia were different (anterior: 153.2 ± 39.3 µm; medial: 128.4 ± 24.7 µm; lateral: 154 ± 28.9 µm; and posterior: 148.8 ± 33.2 µm) as were those of the deep fascia (anterior: 556.8 ± 176.2 µm; medial: 820.4 ± 201 µm; lateral: 1112 ± 237.9 µm; and posterior: 730.4 ± 186.5 µm). The US scans showed a clear picture of the superficial adipose tissue, the superficial fascia, and the deep adipose tissue, as well as the deep fasciae. The epimysial and aponeurotic fasciae of only some topographic areas could be independently identified. The US imaging findings confirmed that the superficial and deep fascia have different thicknesses, and they showed that the US measurements were always larger with respect to those produced by histological analysis (p < 0.001) probably due to shrinkage during the processing. The posterior region (level 1) of the superficial fascia had, for example, a mean thickness of 0.56 ± 0.12 mm at US, while the histological analysis showed that it was 148.8 ± 33.2 µm. Showing a similar pattern, the thickness of the deep fascia was as follows: 1.64 ± 0.85 mm versus 730.4 ± 186.5 µm. Study results have confirmed that US can be considered a valid, non-invasive instrument to evaluate the fascial layers. In any event, there is a clear need for a set of standardised protocols since the thickness of the fascial layers of different parts of the human body varies and the data obtained using inaccurate reference points are not reproducible or comparable. Given the inconsistent terminology used to describe the fascial system, it would also be important to standardise the terminology used to define its parts. The difficulty in distinguishing between the epimysial and aponeurotic/deep fascia can also impede data interpretation.