Proteomics analysis of deep fascia in acute compartment syndrome.

Acute compartment syndrome (ACS) is a syndrome in which local circulation is affected due to increased pressure within the compartment. We previously found in patients with calf fractures, the pressure of fascial compartment could be sharply reduced upon the appearance of tension blisters. Deep fascia, as the important structure for compartment, might play key role in this process. Therefore, the aim of the present study was to examine the differences in gene profile in deep fascia tissue in fracture patients of the calf with or without tension blisters, and to explore the role of fascia in pressure improvement in ACS. Patients with lower leg fracture were enrolled and divided into control group (CON group, n = 10) without tension blister, and tension blister group (TB group, n = 10). Deep fascia tissues were collected and LC-MS/MS label-free quantitative proteomics were performed. Genes involved in fascia structure and fibroblast function were further validated by Western blot. The differentially expressed proteins were found to be mainly enriched in pathways related to protein synthesis and processing, stress fiber assembly, cell-substrate adhesion, leukocyte mediated cytotoxicity, and cellular response to stress. Compared with the CON group, the expression of Peroxidasin homolog (PXDN), which promotes the function of fibroblasts, and Leukocyte differentiation antigen 74 (CD74), which enhances the proliferation of fibroblasts, were significantly upregulated (p all <0.05), while the expression of Matrix metalloproteinase-9 (MMP9), which is involved in collagen hydrolysis, and Neutrophil elastase (ELANE), which is involved in elastin hydrolysis, were significantly reduced in the TB group (p all <0.05), indicating fascia tissue underwent microenvironment reconstruction during ACS. In summary, the ACS accompanied by blisters is associated with the enhanced function and proliferation of fibroblasts and reduced hydrolysis of collagen and elastin. The adaptive alterations in the stiffness and elasticity of the deep fascia might be crucial for pressure release of ACS.

Organ dependency on fascia connective tissue.

Fascia is a specialized connective tissue system that encapsulates and interconnects between tissues and organs throughout the body. The fascia system regulates pain sensation, organ inflammation, trauma, and fibrotic diseases. This mini-review summarizes recent findings from animal models, which reveal the inter-dependency between tissues/organs and the fascia system. Special mechanisms are explored of fascia response to skin inflammatory processes and fibrotic microenvironments in trauma models. We highlight the functionally diverse communities of its fascia-born fibroblasts and the significance of their stage-specific differentiation and communication to disease progression. Understanding the molecular mechanisms and cellular processes within the fascia microenvironment may serve as a basis for future clinical translation.

Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy.

BACKGROUND: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown. PURPOSE: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms. STUDY DESIGN: Descriptive laboratory study. METHODS: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue. RESULTS: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17). CONCLUSION: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life. CLINICAL RELEVANCE: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Proteomic Analysis of Dupuytren's Contracture-Derived Sweat Glands Revealed the Synthesis of Connective Tissue Growth Factor and Initiation of Epithelial-Mesenchymal Transition as Major Pathogenetic Events.

Dupuytren's contracture (DC) is a chronic and progressive fibroproliferative disorder restricted to the palmar fascia of the hands. Previously, we discovered the presence of high levels of connective tissue growth factor in sweat glands in the vicinity of DC nodules and hypothesized that sweat glands have an important role in the formation of DC lesions. Here, we shed light on the role of sweat glands in the DC pathogenesis by proteomic analysis and immunofluorescence microscopy. We demonstrated that a fraction of sweat gland epithelium underwent epithelial-mesenchymal transition illustrated by negative regulation of E-cadherin. We hypothesized that the increase in connective tissue growth factor expression in DC sweat glands has both autocrine and paracrine effects in sustaining the DC formation and inducing pathological changes in DC-associated sweat glands.

Effect of nanoparticle-mediated delivery of SFRP4 siRNA for treating Dupuytren disease.

Dupuytren disease (DD) is a progressive fibrous proliferative disease. It invades the palmar aponeurosis and extends to the finger fascia, eventually leading to flexion contracture of the metacarpophalangeal or interphalangeal joint. At present, surgical resection and the local injection of collagenase are the main methods for the treatment of DD, but postoperative complications and high recurrence rates often occur. Bioinformatics analysis showed that the increased expression of SFRP4 protein was closely related to the incidence of DD. Persistent and effective inhibition of SFRP4 expression may be a promising treatment for DD. We prepared SFRP4 siRNA/nanoparticle complexes (si-SFRP4) and negative siRNA/nanoparticle complexes (NC) and applied them in vitro and in vivo. Flow cytometry analysis showed that si-SFRP4 could be successfully transfected into DD cells. MTT and EdU staining assays showed that the OD values and percentage of EdU-positive cells in the si-SFRP4 group were significantly lower than those in the NC group. Scratch tests showed that the wound healing rate of the si-SFRP4 group was lower than that of the NC group, and the difference was statistically significant. The expression of SFRP4 and α-SMA protein in the si-SFRP4 group significantly decreased in both DD cells and xenografts. Compared with the NC group, the xenograft quality of the si-SFRP4 group was significantly reduced. Masson's trichrome staining showed that the collagen and fibrous cells in the si-SFRP4 group were more uniform, slender, parallel and regular. The above experimental results suggest that the proliferation and metabolism of palmar aponeurosis cells and the quality of metacarpal fascia xenografts were both significantly decreased. We speculated that nanoparticle-mediated SFRP4 siRNA can be used as a potential new method for the treatment of DD.

Pathophysiological and Therapeutic Roles of Fascial Hyaluronan in Obesity-Related Myofascial Disease.

Myofascial disease is an important complication associated with obesity and one of the leading causes of physical disability globally. In the face of limited treatment options, the burden of myofascial disorders is predicted to increase along with the escalating prevalence of obesity. Several pathological processes in obesity contribute to modifications in fascial extracellular matrix mechanical and biological properties and functions. Changes in adipose tissue metabolism, chronic inflammatory phenotype, oxidative stress, and other mechanisms in obesity may alter the physiochemical and biomechanical properties of fascial hyaluronan. Understanding the pathophysiological importance of hyaluronan and other components of the fascial connective tissue matrix in obesity may shed light on the etiology of associated myofascial disorders and inform treatment strategies. Given its unique and favorable pharmacological properties, hyaluronan has found a broad range of clinical applications, notably in orthopedic conditions such as osteoarthritis and tendinopathies, which share important pathophysiological mechanisms implicated in myofascial diseases. However, while existing clinical studies uniformly affirm the therapeutic value of hyaluronan in myofascial disorders, more extensive studies in broader pharmacological and clinical contexts are needed to firmly validate its therapeutic adaptation.

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.

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia.

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.

Hyaluronan and the Fascial Frontier.

The buzz about hyaluronan (HA) is real. Whether found in face cream to increase water volume loss and viscoelasticity or injected into the knee to restore the properties of synovial fluid, the impact of HA can be recognized in many disciplines from dermatology to orthopedics. HA is the most abundant polysaccharide of the extracellular matrix of connective tissues. HA can impact cell behavior in specific ways by binding cellular HA receptors, which can influence signals that facilitate cell survival, proliferation, adhesion, as well as migration. Characteristics of HA, such as its abundance in a variety of tissues and its responsiveness to chemical, mechanical and hormonal modifications, has made HA an attractive molecule for a wide range of applications. Despite being discovered over 80 years ago, its properties within the world of fascia have only recently received attention. Our fascial system penetrates and envelopes all organs, muscles, bones and nerve fibers, providing the body with a functional structure and an environment that enables all bodily systems to operate in an integrated manner. Recognized interactions between cells and their HA-rich extracellular microenvironment support the importance of studying the relationship between HA and the body's fascial system. From fasciacytes to chronic pain, this review aims to highlight the connections between HA and fascial health.

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.

Connexin43 gap junction drives fascia mobilization and repair of deep skin wounds.

Deep and voluminous skin wounds are repaired with scars, by mobilization of fibroblasts and extracellular matrix from fascia, deep below the skin. The molecular trigger of this novel repair mechanism is incompletely understood. Here we reveal that the gap junction alpha-1 protein (Connexin43, Cx43) is the key to patch repair of deep wounds. By combining full-thickness wound models with fibroblast lineage specific transgenic lines, we show Cx43 expression is substantially upregulated in specialized fibroblasts of the fascia deep beneath the skin that are responsible for scar formation. Using live imaging of fascia fibroblasts and fate tracing of the fascia extracellular matrix we show that Cx43 inhibition disrupts calcium oscillations in cultured fibroblasts and that this inhibits collective migration of fascia EPFs necessary to mobilize fascia matrix into open wounds. Cell-cell communication through Cx43 thus mediates matrix movement and scar formation, and is necessary for patch repair of voluminous wounds. These mechanistic findings have broad clinical implications toward treating fibrosis, aggravated scarring and impaired wound healing.

Injury triggers fascia fibroblast collective cell migration to drive scar formation through N-cadherin.

Scars are more severe when the subcutaneous fascia beneath the dermis is injured upon surgical or traumatic wounding. Here, we present a detailed analysis of fascia cell mobilisation by using deep tissue intravital live imaging of acute surgical wounds, fibroblast lineage-specific transgenic mice, and skin-fascia explants (scar-like tissue in a dish - SCAD). We observe that injury triggers a swarming-like collective cell migration of fascia fibroblasts that progressively contracts the skin and form scars. Swarming is exclusive to fascia fibroblasts, and requires the upregulation of N-cadherin. Both swarming and N-cadherin expression are absent from fibroblasts in the upper skin layers and the oral mucosa, tissues that repair wounds with minimal scar. Impeding N-cadherin binding inhibits swarming and skin contraction, and leads to reduced scarring in SCADs and in animals. Fibroblast swarming and N-cadherin thus provide therapeutic avenues to curtail fascia mobilisation and pathological fibrotic responses across a range of medical settings.

Sensitivity of the Fasciae to the Endocannabinoid System: Production of Hyaluronan-Rich Vesicles and Potential Peripheral Effects of Cannabinoids in Fascial Tissue.

The demonstrated expression of endocannabinoid receptors in myofascial tissue suggested the role of fascia as a source and modulator of pain. Fibroblasts can modulate the production of the various components of the extracellular matrix, according to type of stimuli: physical, mechanical, hormonal, and pharmacological. In this work, fascial fibroblasts were isolated from small samples of human fascia lata of the thigh, collected from three volunteer patients (two men, one woman) during orthopedic surgery. This text demonstrates for the first time that the agonist of cannabinoid receptor 2, HU-308, can lead to in vitro production of hyaluronan-rich vesicles only 3-4 h after treatment, being rapidly released into the extracellular environment. We demonstrated that these vesicles are rich in hyaluronan after Alcian blue and Toluidine blue stainings, immunocytochemistry, and transmission electron microscopy. In addition, incubation with the antagonist AM630 blocked vesicles production by cells, confirming that release of hyaluronan is a cannabinoid-mediated effect. These results may show how fascial cells respond to the endocannabinoid system by regulating and remodeling the formation of the extracellular matrix. This is a first step in our understanding of how therapeutic applications of cannabinoids to treat pain may also have a peripheral effect, altering the biosynthesis of the extracellular matrix in fasciae and, consequently, remodeling the tissue and its properties.

Sensitivity of the fasciae to sex hormone levels: Modulation of collagen-I, collagen-III and fibrillin production.

Although it is now recognized that women suffer from myofascial pain to a greater extent than men, and that the muscular fasciae can respond to hormonal stimuli, thanks to the expression of sex hormone receptors, how the fasciae can modify their structure under hormonal stimulation is not clear. In this work, an immunocytochemical analysis of collagen-I, collagen-III and fibrillin were carried out on fibroblasts isolated from human fascia lata after in vitro treatment with various levels of sex hormones ß-estradiol and/or relaxin-1, according to the phases of a woman's period (follicular, periovulatory, luteal, post-menopausal phases and pregnancy). This study demonstrates for the first time that fascial cells can modulate the production of some components of the extracellular matrix according to hormone levels, when treated with ß-estradiol: collagen-I falls from 6% of positivity in the follicular phase to 1.9 in the periovulatory phase. However, after the addition of relaxin-1 to the cell culture, the production of extracellular matrix decreased and remained at the same level (1.7% of collagen-I, at both follicular and periovulatory levels of hormones). These results confirm the antifibrotic function of relaxin-1, thanks to its ability to reduce matrix synthesis. They are also a first step in our understanding of how some hormonal dysfunctions in women can cause a dysregulation of extracellular matrix production in fasciae.

Blue light irradiation and its beneficial effect on Dupuytren's fibroblasts.

Dupuytren's contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm2, continuous radiance, spot size 10-12 cm2) as well as the molecular mechanism mediating this effect. It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren's fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren's disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.

Osteoinduction within BMP-2 transduced muscle tissue fragments with and without a fascia layer: implications for bone tissue engineering.

Bone can be engineered in vivo by implantation of gene-activated muscle tissue fragments. This expedited approach may be further improved by use of muscle tissue with attached fascia. The aim of this in vitro study was to provide an in depth comparison of the osteogenic differentiation capacity of muscle alone and muscle with fascia after BMP-2 transduction. Skeletal muscle tissue from rats was cut into pieces with and without a fascia layer on the surface. Adenoviral BMP-2 or GFP vectors were used for transduction. Osteogenic differentiation within the tissue fragments was evaluated and compared by qRT-PCR, alizarin red S staining, histomorphometry and immunohistology. Transduction efficiency and level of transgene expression were higher for muscle with fascia than muscle alone. Transduction with BMP-2 led to a significant upregulation of bone marker genes, proteins, and calcium deposition in both groups. Interestingly, histological evaluation revealed that osteoinduction did not occur within the fascia layer itself. The upregulation of bone marker genes in muscle with fascia was significantly lower after 2 weeks but similar after 4 weeks of in vitro culture in comparison to muscle alone. The fascia layer led to higher transduction efficiency and enhanced BMP-2 expression. Despite fascia's lower capacity for osteogenic differentiation, muscle implants may benefit from the fascia layer by the improved ability to deliver BMP-2. The presented data may contribute to the development of a novel, cost-effective, single-surgery bone engineering technology and encourage the evaluation of the osteoregenerative potential of muscle with fascia in an animal model.

Variations in contents of hyaluronan in the peritumoral micro-environment of human chondrosarcoma.

A concept consolidated in recent years is that tumor growth depends to a great extent on the micro-environment surrounding the tumor, which has a fundamental role in tumor progression and in determining the effectiveness of therapies. Our analysis focuses on chondrosarcoma, the second primary malignant bone tumor, resistant to both chemotherapeutic and radiation therapy. We quantified hyaluronan, one of the main components of the extracellular matrix, with the aim of comparing its amount in the connective tissue surrounding the tumor with intra-tumoral tissue and healthy fascia of the same anatomic district, viewed as a health control. We demonstrate that hyaluronan increased significantly in the peritumoral stroma compared with the healthy fascia, which showed an average amount according to the physical characteristics of body districts by a mean value of 26.9 µg/g. In the peritumoral stroma, the mean hyaluronan content reached 132.6 µg/g (mean value of 63.2 µg/g). The p-value was less than 0.01, showing a highly significant statistical difference. Surprisingly, no significant differences were detected as a function of age, gender, or tumor grade. The levels of hyaluronan were comparable in peritumoral and tumor tissues, although there were differences depending on the state of necrosis. In addition, data on the expression of hyaluronic acid synthetase showed a decrease of about 50% in peritumoral and tumor tissues, indicating alterations in hyaluronan turnover and synthesis. This work demonstrates a variation in hyaluronan contents around the chondrosarcoma, likely correlated with the aggressiveness and resistance to chemotherapy of this tumors. Statement of Clinical Significance: Deeper knowledge about the composition of the peritumoral stroma, rich in extracellular matrix, will enhance better study and understanding of the metastatic potential of tumors and their prognostic indices. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:503-509, 2019.

The effect of the volume of supra-inguinal injected solution on the spread of the injectate under the fascia iliaca: a preliminary study.

The fascia iliaca compartment is the compartment confined by the fascia iliaca (FI) and a muscular layer formed by the iliac- and psoas muscle. This compartment creates a virtual tunnel that contains the femoral nerve (FN), the obturator nerve (ON), and the lateral femoral cutaneous nerve (LFCN) of the lumbar plexus. In this pilot study, we aimed to determine the suggested volume needed to reach the three target nerves of the lumbar plexus (FN, ON, and LFCN) with a single-injection ultrasound-guided supra-inguinal fascia iliaca compartment (S-FICB). A computer tomography (CT scan)-guided step-up/step-down sequence was used to determine the suggested injection volume to target all three nerves. Subsequently, an anatomist blinded for the injected volume and CT findings, dissected the cadavers, and evaluated the spread of dye underneath the fascia iliaca. In total, seven pelvic areas of four cadavers were evaluated on CT scan and dissected. Distribution of dye underneath the FI in relation to the FN, ON, and the LFCN was recorded in all dissected cadavers. Combining CT and dissection findings, the suggested volume to reach the FN, ON, and LFCN with an S-FICB was 40 mL.