Quantification of hyaluronan in human fasciae: variations with function and anatomical site.

Recently, alterations in fascial gliding-like movement have been invoked as critical in the etiology of myofascial pain. Various methods have been attempted for the relief of this major and debilitating clinical problem. Paramount have been attempts to restore correct gliding between fascial layers and the movement over bone, joint, and muscular structures. One of the key elements that underlies such fascial movement is hyaluronan. However, until now, the precise content of hyaluronan within fasciae has been unknown. This study quantifies for the first time the hyaluronan content of human fascial samples obtained from a variety of anatomic sites. Here, we demonstrate that the average amount varies according to anatomic site, and according to the different kinds of sliding properties of the particular fascia. For example, the fascia lata has 35 µg of hyaluronan per gram of tissue, similar to that of the rectus sheath (29 µg g-1 ). However, the types of fascia adherent to muscle contain far less hyaluronan: 6 µg g-1 in the fascia overlying the trapezius and deltoid muscles. In the fascia that surrounds joints, the hyaluronan increases to 90 µg g-1 , such as in the retinacula of the ankle, where greater degrees of movement occur. Surprisingly, no significant differences were detected at any site as a function of age or sex (P-value > 0.05, t-test) with the sole exception of the plantar fascia. This work can provide a better understanding of the role of hyaluronan in fascia. It will facilitate a better comprehension of the modulation of the hyaluronan-rich layer that occurs in relation to the various conditions that affect fascia, and the diverse factors that underlie the attendant pathologies.

Transanal ileoproctostomy is feasible in human cadavers.

AIM: An ileorectal bypass performed entirely through a transanal route has recently been described in an animal model. The present study aimed to demonstrate its technical feasibility in four human cadavers. METHOD: A transanal endoscopic microsurgery (TEM) device and endoscopic instruments were used. The principal steps of the procedure included insertion of the TEM device, rectostomy above the peritoneal reflection, peritoneoscopy using a standard gastroscope and delivery of the small bowel through the proctostomy to perform an anastomosis. RESULTS: The procedure was successfully completed using transanal access in all cases. The mean procedure time was 90 min. The bypass was patent, and the anastomosis between the intraperitoneal rectum and the terminal ileum was leakproof. CONCLUSION: Transanal ileoproctostomy is technically feasible in human cadavers. The procedure may become an alternative to stoma formation in selected patients with colonic obstruction.

Development and aging are oxygen-dependent and correlate with VEGF and NOS along life span.

During development and aging, vascular remodeling represents a critical adaptive response to modifications in oxygen supply to tissues. Hypoxia inducible factor (HIF) has a crucial role and is modulated by oxygen levels, with an age-dependent response in neonates, adult, and aged people. ROS are generated under hypoxic conditions and the accumulation of free radicals during life reduces the ability of tissues to their removal. In this immunohistochemical study we investigated the presence and localization of VEGF and iNOS in human carotid bodies (CB) sampled at autopsy from three children (mean age - 2 years), four adult young subjects (mean age - 44.3 years), and four old subjects (mean age - 67.3 years). VEGF immunoreactivity was significantly enhanced in CB tissues from the children (7.2 ± 1.2%) and aged subjects (4.7 ± 1.7%) compared with the young adults (1.4 ± 0.7%). On the other hand, iNOS immunoreactivity was enhanced in CB tissues from the children (0.4 ± 0.04%) and young adult subjects (0.3 ± 0.02%) compared with the old subjects (0.2 ± 0.02%). Prevention of oxygen desaturation, reducing all causes of hypoxemia from neonatal life to aging would decrease the incidence of diseases in the elderly population with lifespan extension.

Cytoglobin and neuroglobin in the human brainstem and carotid body.

The aim of the present study was to evaluate the presence of Neuroglobin (Ngb) and Cytoglobin (Cygb) in the solitary tract nucleus (STN) and in the carotid body of human subjects. Transverse serial sections of formalin-fixed, paraffin-embedded brainstems, taken from six subjects, were investigated. Ngb and Cygb are expressed in both the structures. Differences in expression of Ngb and Cygb among dorsal and ventral area of the STN may be related to their different functions and different metabolic demands. Because the STN plays an important role in the processing of cardiovascular and respiratory reflex inputs, Ngb and Cygb may play an integrative central modulatory action for the two systems.

Tullio Terni (1888-1946): The "column" of spinal cardiovascular regulation.

Tullio Terni (1888-1946) was a brilliant anatomist in the School of Medicine of Padova, Italy. He was a versatile scientist who gave fundamental and pioneering contributions in descriptive and experimental cytology, human and comparative morphogenesis, neuroanatomy, embryology and teratology, and regenerative biology. His most famous discovery, which bears his name, is the so-called "Terni's column." In embryos of chickens, he described the existence in the thoracolumbar region of the spinal cord of a preganglionic nervous center, constituting a longitudinal column of nervous cells between the first thoracic and the second lumbar segments. Tullio Terni embodied the ideal of free science without geographic boundaries. He used cutting-edge tools, demonstrating his very current approach. Terni studied the organization of tissues and organs and the spatial arrangement and the physical state of the tissues of living systems. He also practiced experimental embryology, which formed the basis of modern techniques in organ transplantation. Moreover, he studied multiple species in order to compare multiple organisms. Terni was a multifaceted scientist.

Sulci of the liver found after death: Their nature and potential teaching value.

Accessory sulci of the liver are more commonly found after death than in life, raising questions as to their causation and possible classification. We have analyzed a group of 180 livers sampled from un-embalmed (96) and embalmed cadavers (84). In un-embalmed cadavers, no accessory sulci were found on the diaphragmatic surface in 58 cases. Diaphragmatic sulci were found in the right lobe of 38 livers. When removed from the abdominal cavity and placed flat on the examination table (the "bench position") all 58 livers without sulci appreciable in the abdominal cavity showed the appearance of two sulci. The first ran from the right side of the inferior vena cava (IVC), curving anteriorly to the inferior border of the liver, at a point midway between the right extremity of the inferior border and the gallbladder fossa, concave towards the left. The second sulcus ran from the left side of the IVC, curving anteriorly to reach the inferior border of the liver at the level of the gallbladder fossa, concave towards the right. With progressive side-to-side manual compression, the sulci on the diaphragmatic surface become more evident. Division of the hepatic parenchyma along the two sulci exposed the right and middle hepatic veins respectively in more than 90% of cases. In embalmed cadavers, 24 livers showed antero-posterior sulci in the superior surface, visible and palpable on the liver examined in situ. When the livers with sulci had been removed from the abdomen for further examination, the appearance of the superior surface did not change. In a removed liver, accessory sulci can be divided into true, "diaphragmatic," sulci and "false" sulci due to the position of the free liver on the examination table. The "false" sulci may be considered as further morphological evidence of the functional anatomical division of the liver. Their demonstration may also be useful in teaching its topographical and surgical anatomy.

Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

Hyaluronan within fascia in the etiology of myofascial pain.

The layers of loose connective tissue within deep fasciae were studied with particular emphasis on the histochemical distribution of hyaluronan (HA). Samples of deep fascia together with the underlying muscles were taken from neck, abdomen and thigh from three fresh non-embalmed cadavers. Samples were stained with hematoxylin-eosin, Azan-Mallory, Alcian blue and a biotinylated HA-binding protein specific for HA. An ultrasound study was also performed on 22 voluntary subjects to analyze the thickness of these deep fasciae and their sublayers. The deep fascia presented a layer of HA between fascia and the muscle and within the loose connective tissue that divided different fibrous sublayers of the deep fascia. A layer of fibroblast-like cells that stained prominently with Alcian blue stain was observed. It was postulated that these are cells specialized for the biosynthesis of the HA-rich matrix. These cells we have termed "fasciacytes", and may represent a new class of cells not previously recognized. The ultrasound study highlighted a mean thickness of 1.88 mm of the fascia lata, 1.68 mm of the rectus sheath, and 1.73 mm of the sternocleidomastoid fascia. The HA within the deep fascia facilitates the free sliding of two adjacent fibrous fascial layers, thus promoting the normal function associated with the deep fascia. If the HA assumes a more packed conformation, or more generally, if the loose connective tissue inside the fascia alters its density, the behavior of the entire deep fascia and the underlying muscle would be compromised. This, we predict, may be the basis of the common phenomenon known as "myofascial pain."

Investigation of the mechanical properties of the plantar aponeurosis.

INTRODUCTION: The aim of this work was to obtain a preliminary investigation of the mechanical properties of the human plantar aponeurosis based on regional observation, in order to rationally plan a subsequent larger experimental campaign and develop suited constitutive models to characterize the mechanical response of this tissue. MATERIALS AND METHODS: Different in vitro mechanical tests were developed on eleven samples taken from the plantar aponeurosis of human cadaver (man, age 78 years). The samples were tested along the distal-proximal direction. Range of elasticity of the tissue, development of damage phenomena and stress relaxation at different levels of strain were evaluated. RESULTS: The strength of the tissue was found in the order of that proposed in previous works, with peak stress of about 12.5 MPa. The compliance of the plantar aponeurosis was in line with in vivo evaluation. A softening behaviour appeared for tensile strain larger than 12%. In relaxation tests, the stress was reduced of 35-40% in 120 s. The percentage stress relaxation was found independent on the level of the applied strain. DISCUSSION: The evaluation of the mechanical characteristics is fundamental for a subsequent development of numerical models of the plantar aponeurosis. Such approach is helpful to understand its response to overuse, but also to understand the clinical results of different manual and physical therapies that use warm, pressure or stretch to modify this tissue.

3D reconstruction of the crural and thoracolumbar fasciae.

PURPOSE: To create computerized three-dimensional models of the crural fascia and of the superficial layer of the thoracolumbar fascia. METHODS: Serial sections of these two fasciae, stained with Azan-Mallory, van Gieson and anti-S100 antibody stains, were recorded. The resulting images were merged (Image Zone 5.0 software) and aligned (MatLab Image Processing Toolkit). Color thresholding was applied to identify the structures of interest. 3D models were obtained with Tcl/Tk scripts and Paraview 3.2.1 software. From these models, the morphometric features of these fasciae were evaluated with ImageJ. RESULTS: In the crural fascia, collagen fibers represent less than 20% of the total volume, arranged in three distinct sub-layers (mean thickness, 115 µm), separated by a layer of loose connective tissue (mean thickness, 43 µm). Inside a single sub-layer, all the fibers are parallel, whereas the angle between the fibers of adjacent layers is about 78°. Elastic fibers are less than 1%. Nervous fibers are mostly concentrated in the middle layer. The superficial layer of the thoracolumbar fascia is also formed of three thinner sub-layers, but only the superficial one is similar to the crural fascia sub-layers, the intermediate one is similar to a flat tendon, and the deep one is formed of loose connective tissue. Only the superficial sub-layer has rich innervation and a few elastic fibers. DISCUSSION: Computerized three-dimensional models provide a detailed representation of the fascial structure, for better understanding of the interactions among the different components. This is a fundamental step in understanding the mechanical behavior of the fasciae and their role in pathology.

Neurotensin receptor 1 immunoreactivity in the peripheral ganglia and carotid body.

In the present study we investigated, through immunohistochemistry, the presence and location of neurotensin receptor 1 (NTR1) in the peripheral ganglia and carotid body of 16 humans and 5 rats. In both humans and rats, NTR1 immunostained ganglion cells were found in superior cervical ganglia (57.4+/-11.6% and 72.4+/-11.4%, respectively, p0.05), enteric ganglia (51.9+/-10.4% and 64.6+/-6.1, p<0.05), sensory ganglia (69.2+/-10.7% and 73.0+/-13.1%, p>0.05) and parasympathetic ganglia (52.1+/-14.1% and 59.4+/-14.0%, p>0.05), supporting a modulatory role for NT in these ganglia. Positivity was also detected in 45.6+/-9.2% and 50.8+/-6.8% of human and rat type I glomic cells, respectively, whereas type II cells were negative. Our findings suggest that NT produced by type I cells acts in an autocrine or paracrine way on the same cell type, playing a modulatory role on chemoception.

Anatomic distribution of apoptosis in medulla oblongata of infants and adults.

The aim of the study was to evaluate the distribution of apoptosis in the medullary nuclei of infants and adults who died of hypoxic-ischaemic injury. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) in brainstems from 22 adults (7 subjects who died of opiate intoxication, 15 who died of other hypoxic-ischaemic injury) and 10 infants. The nuclei examined included the hypoglossal, dorsal motor nucleus of the vagus, nucleus tractus solitarii, nucleus of the spinal trigeminal tract, cuneate, vestibular and inferior olivary nuclei. A morphometric analysis with the optical disector method was performed to calculate the mean percentages (+/- standard deviation) of TUNEL-positive neuronal and glial cells for the sample populations. Opiate deaths did not have higher apoptotic indices than other adult hypoxic-ischaemic deaths. Statistically significant differences between adults and infants were found in the neuronal apoptotic indices of the cuneate (28.2 +/- 16.3% vs. 6.9 +/- 8.7%), vestibular (24.7 +/- 15.0% vs. 11.3 +/- 11.4%), nucleus tractus solitarii (11.2 +/- 11.2% vs. 2.3 +/- 2.4%), dorsal motor nucleus of the vagus (6.8 +/- 8.5% vs. 0.1 +/- 0.2%) and hypoglossal (6.6 +/- 5.7% vs. 0.1 +/- 0.2%), indicating higher resistance of the neuronal populations of these infant medullary nuclei to terminal hypoxic-ischaemic injury or post-mortem changes. Differences in neuronal apoptotic index were also statistically significant among nuclei, suggesting differential characteristics of survival. Nuclei with higher neuronal apoptotic indices were the cuneate, vestibular and nucleus of the spinal trigeminal tract, which are located in the lateral medullary tegmentum and share the same vascular supply from the posterior inferior cerebellar artery.

The gracilis muscle and its use in clinical reconstruction: an anatomical, embryological, and radiological study.

The gracilis muscle is used widely in reconstructive surgery, as a pedicled or as a free microsurgical flap, for soft tissue coverage or as a functioning muscle transfer. Many studies, based on cadaver dissections, have focused on the vascular anatomy of the gracilis muscle and provided different data about the number, origin, and caliber of its vascular pedicles. Computed tomographic (CT) angiography of both thighs of 40 patients (35 males and 5 females, mean age: 63 years) have been analyzed to provide a detailed anatomical description of the arterial supply of the gracilis muscle. The gracilis muscle had a mean length of 41 +/- 2.1 cm. The principal pedicle enters the gracilis muscle at a mean distance (+/-SD) of 10 +/- 1 cm from the ischiopubic attachment of the muscle. Its caliber shows a mean value of 2.5 +/- 0.5 mm, and it is statistically larger when originating directly from the deep femoral artery (45%) than from its muscular branch supplying the adductors, i.e., the "artery to the adductors" (46%) (P < 0.01). A significant correlation between the caliber of the artery of the main pedicle and the volume of the gracilis muscle was found (P < 0.01). The mean number of distal accessory pedicles is 1.8 (range, 1-4,) and the artery of the first of these pedicles shows a mean caliber of 2.0 mm. There is no correlation between either the number or the caliber of the artery of the accessory pedicles and the volume of the gracilis muscle. CT angiography, providing detailed images of the muscular and vascular structures of the thigh of each patient, could be a useful preoperative study for the reconstructive surgeon. It would allow a personalized planning of a gracilis flap, reducing the risk of iatrogenic damage.

Tendinous muscular insertions onto the deep fascia of the upper limb. First part: anatomical study.

We examined 30 upper limbs in order to study the tendinous muscular insertions into the deep fascia and to verify whether they have a specific anatomical arrangement and to measure their resilience to traction. We have found that the fascia receives many tendinous muscular insertions, which are always present and exhibit a constant anatomical structure. In particular, the pectoralis major fascia always continues with the brachial fascia in two distinct ways: the fascia overlying the clavicular part of pectoralis major had an expansion towards the anterior brachial fascia, whereas the fascia covering its costal part extended into the medial brachial fascia and the medial intermuscular septum. The lacertus fibrosus was also composed by two groups of fibres: the main group was oriented downwards and medially, the second group longitudinally. The palmaris longus opened out into a fan-shape in the palm of the hand and sent some tendinous expansions to the flexor retinaculum and fascia overlying the thenar eminence muscles. In the posterior region of the arm, the fascia of the latissimus dorsi sent a fibrous lamina to the triceps brachial fascia. The triceps tendon inserted partially into the antebrachial fascia, while the extensor carpi ulnaris sent a tendinous expansion to the fascia of the hypothenar eminence. It is hypothesized that the tendinous muscular insertions maintain the fascia at a basal tension and create myofascial continuity between the different muscles actuating flexion and extension of the upper limb, stretching the fascia in different ways according to the different motor directions.

Anatomy of the deep fascia of the upper limb. Second part: study of innervation.

Analysis of specimens taken from different areas of the deep fascia in 20 upper limbs was made in order to establish which kind of nerve fibres and endings are present in the deep muscular fascia. The flexor retinaculum and the lacertus fibrosus were also evaluated because they are anatomically hardly separable from the deep muscular fascia, although they have different functions. In particular, specimens were taken at the level of: (a) the expansion of pectoralis major onto the bicipital fascia, (b) the middle third of the brachial fascia, (c) the lacertus fibrosus, (d) the middle third of the antebrachial fascia, (e) the flexor retinaculum. This study demonstrated an abundant innervation of the fascia consisting in both free nerve endings and encapsulated receptors, in particular, Ruffini and Pacini corpuscles. However, differences in innervation were verified: the flexor retinaculum was resulted the more innervated element whilst lacertus fibrosus and the pectoralis major expansion the less innervated. These results suggest that the retinaculum has more a perceptive function whereas the tendinous expansions onto the fascia have mostly a mechanical role in the transmission of tension. The hypothesis that the fascia plays an important role in proprioception, especially dynamic proprioception, is therefore advanced. In fact, the fascia is a membrane that extends throughout the whole body and numerous muscular expansions maintain it in a basal tension. During a muscular contraction these expansions could also transmit the effect of the stretch to a specific area of the fascia, stimulating the proprioceptors in that area.

Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles.

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

The triangles of Grynfeltt and Petit and the lumbar tunnel: an anatomo-radiologic study.

PURPOSE: Lumbar hernias are protrusions of intra-abdominal contents classically through the superior (Grynfeltt) and inferior (Petit) lumbar triangles. The anatomy of the triangles is variable and quantitative data are few. No radiological data on the anatomy of the triangles are available. METHODS: Fifty computed tomography angiography of the upper abdomen (M25, F25, mean age 72.5-year-old) were analyzed. The dimensions and the contents of the lumbar triangles were analyzed. The characteristics of the space between the two triangles were also documented. RESULTS: The superior lumbar triangle showed a mean surface area of 5.10 ± 2.6 cm2. In the area of the triangle, the 12th intercostal pedicle and the 1st lumbar branches of the iliolumbar vessels were found in 42 and 46 %, respectively. The inferior lumbar triangle had a mean surface of area 18.7 ± 8.4 cm2. In this area, the 2nd, 3rd, and 4th lumbar branches were found in 9, 67, and 8 %, respectively. On oblique coronal images, a direct tunnel between the superior and the inferior lumbar triangles was found, showing an oblique course, with a postero-anterior direction (mean length 36.5 ± 5.8 mm, mean caliber 7.4 ± 3.1 mm). CONCLUSIONS: Among the anatomical factors of weakening of the abdominal wall, the course of branches of the lumbar vessels was documented not only in the superior but also in the inferior lumbar triangle. A real musculoaponeurotic tunnel between the superior and the inferior lumbar triangles located in the oblique coronal plane was found, that could play a role in the development of incarceration or strangulation of lumbar hernias.

Hormone receptor expression in human fascial tissue.

Many epidemiologic, clinical, and experimental findings point to sex differences in myofascial pain in view of the fact that adult women tend to have more myofascial problems with respect to men. It is possible that one of the stimuli to sensitization of fascial nociceptors could come from hormonal factors such as estrogen and relaxin, that are involved in extracellular matrix and collagen remodeling and thus contribute to functions of myofascial tissue. Immunohistochemical and molecular investigations (real-time PCR analysis) of relaxin receptor 1 (RXFP1) and estrogen receptor-alpha (ERα) localization were carried out on sample of human fascia collected from 8 volunteers patients during orthopedic surgery (all females, between 42 and 70 yrs, divided into pre- and post-menopausal groups), and in fibroblasts isolated from deep fascia, to examine both protein and RNA expression levels. We can assume that the two sex hormone receptors analyzed are expressed in all the human fascial districts examined and in fascial fibroblasts culture cells, to a lesser degree in the post-menopausal with respect to the pre-menopausal women. Hormone receptor expression was concentrated in the fibroblasts, and RXFP1 was also evident in blood vessels and nerves. Our results are the first demonstrating that the fibroblasts located within different districts of the muscular fasciae express sex hormone receptors and can help to explain the link between hormonal factors and myofascial pain. It is known, in fact, that estrogen and relaxin play a key role in extracellular matrix remodeling by inhibiting fibrosis and inflammatory activities, both important factors affecting fascial stiffness and sensitization of fascial nociceptors.