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.

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.

Expression of the endocannabinoid receptors in human fascial tissue.

Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.

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.

ECRG4 expression in normal rat tissues: expression study and literature review.

The Esophageal Cancer Related Gene 4 (ECRG4) is a highly conserved tumour suppressor gene encoding various peptides (augurin, CΔ16 augurin, ecilin, argilin, CΔ16 argilin) which can be processed and secreted. In the present work, we examined ECRG4 expression and location in a wide range of rat organs and reviewed the available literature. ECRG4 mRNA was identified in all examined tissues by quantitative PCR (qPCR). ECRG4 immunoreaction was mainly cytoplasmic, and was detected in heart and skeletal muscles, smooth muscle cells showing only weak reactions. In the digestive system, ECRG4 immunostaining was stronger in the esophageal epithelium, bases of gastric glands, hepatocytes and pancreatic acinar epithelium. In the lymphatic system, immunoreactive cells were detectable in the thymus cortex, lymph node medulla and splenic red pulp. In the central and peripheral nervous systems, different neuronal groups showed different reaction intensities. In the endocrine system, ECRG4 immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei, hypophysis, thyroid and parathyroid glands, adrenal zona glomerularis and medulla and Leydig cells, as well as in follicular and luteal cells of the ovary. In the literature, ECRG4 has been reported to inhibit cell proliferation and increase apoptosis in various cell types. It is down-regulated, frequently due to hypermethylation, in esophageal, prostate, breast and colon cancers, together with glioma (oncosuppressor function), although it is up-regulated in papillary thyroid cancer (oncogenic role). ECRG4 expression is also higher in non-proliferating cells of the lymphatic system. In conclusion, our identification of ECRG4 in many structures suggests the involvement of ECRG4 in the tumorigenesis of other organs and also the need for further research. In addition, on the basis of the location of ECRG4 in neurons and endocrine cells and the fact that it can be secreted, its role as a neurotransmitter/neuromodulator and endocrine factor must be examined in depth in the future.

Comments to the paper "do we really need new medical information about the Turin Shroud?".

Following the interesting arguments raised in a recent letter to the editor, about a paper recently published in this journal, the authors are happy to take a cue from them to clarify some facts that have not been sufficiently treated for space. After a description of the methods used, arguments regarding a blunt trauma on the right shoulder with consequent dislocation, the position of the hands on the pubis with brachial plexus injury, and the wrist nailing that caused retracted thumbs are discussed in detail.

How was the Turin Shroud Man crucified?

As the literature is not exhaustive with reference to the way the Turin Shroud (TS) Man was crucified, and it is not easy to draw significant information from only a "photograph" of a man on a linen sheet, this study tries to add some detail on this issue based on both image processing of high resolution photos of the TS and on experimental tests on arms and legs of human cadavers. With regard to the TS Man hands, a first hypothesis states that the left hand of the TS Man was nailed twice at two different anatomical sites: the midcarpal joint medially to the pisiform between the lunate/pyramidal and capitate/uncinate bones (Destot's space) and the radiocarpal joint between the radio, lunate and scaphoid; also the right hand would have been nailed twice. A second hypothesis, preferred by the authors, states that the hands were nailed only once in the Destot's space with partial lesion of the ulnar nerve and flexion of the metacarpophalangeal joint of the thumbs. With regard to the TS Man feet, the imprint of the sole of the right foot leads to the conclusion that TS Man suffered a dislocation at the ankle just before the nailing. The entrance hole of the nail on the right foot is a few inches from the ankle, and excludes a double nailing. The nail has been driven between the tarsal bones. The TS Man suffered the following tortures during crucifixion: a very serious and widespread causalgia due to total paralysis of the upper right limb (paradoxical causalgia); a nailing of the left wrist with damage to the ulnar nerve; a similar nailing of the right wrist; and a nailing to both feet using one only nail that injured the plantaris medialis nerves. The respiratory limitation was probably not sufficient to cause death by asphyxiation. Also considering the hypovolemia produced by scourging and the many other tortures detectable on the TS, the principal cause of death can be attributed to a myocardial infarction.

An easy-to-handle microfluidic device suitable for immunohistochemical procedures in mammalian cells grown under flow conditions.

Microfluidic, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.

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.

Do we really need new medical information about the Turin Shroud?

Image processing of the Turin Shroud (TS) shows that the Man represented in it has undergone an under glenoidal dislocation of the humerus on the right side and lowering of the shoulder, and has a flattened hand and enophthalmos; conditions that have not been described before, despite several studies on the subject. These injuries indicate that the Man suffered a violent blunt trauma to the neck, chest and shoulder from behind, causing neuromuscular damage and lesions of the entire brachial plexus. The posture of the left claw-hand is indicative of an injury of the lower brachial plexus, as is the crossing of the hands on the pubis, not above the pubis as it would normally be, and are related to traction of the limbs as a result of the nailing to the patibulum. The disappearance of the thumbprints is because of entrainment of the flexor pollicis longus tendons while the nails were driven through the wrists. The blunt chest trauma, which resulted in the body falling forwards, was the direct cause of a lung contusion and haemothorax, confirmed by the post-mortem leakage of clots and serum from the chest caused by the stabbing with the spear, and was a likely cause of cardiac contusion. All the evidence is in favour of the hypothesis that the TS Man is Jesus of Nazareth.

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.

Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine.

Homologous tissues, such as adipose tissue, may be an interesting source of acellular scaffolds, maintaining a complex physiological three-dimensional (3D) structure, to be recellularized with autologous cells. The aim of the present work is to evaluate the possibility of obtaining homologous acellular scaffolds from decellularization of the omentum, which is known to have a complex vascular network. Adult rat and human omenta were treated with an adapted decellularization protocol involving mechanical rupture (freeze-thaw cycles), enzymatic digestion (trypsin, lipase, deoxyribonuclease, ribonuclease) and lipid extraction (2-propanol). Histological staining confirmed the effectiveness of decellularization, resulting in cell-free scaffolds with no residual cells in the matrix. The complex 3D networks of collagen (azan-Mallory), elastic fibers (Van Gieson), reticular fibers and glycosaminoglycans (PAS) were maintained, whereas Oil Red and Sudan stains showed the loss of lipids in the decellularized tissue. The vascular structures in the tissue were still visible, with preservation of collagen and elastic wall components and loss of endothelial (anti-CD31 and -CD34 immunohistochemistry) and smooth muscle (anti-alpha smooth muscle actin) cells. Fat-rich and well vascularized omental tissue may be decellularized to obtain complex 3D scaffolds preserving tissue architecture potentially suitable for recellularization. Further analyses are necessary to verify the possibility of recolonization of the scaffold by adipose-derived stem cells in vitro and then in vivo after re-implantation, as already known for homologus implants in regenerative processes.

Human carotid body neuroglobin, vascular endothelial growth factor and inducible nitric oxide synthase expression in heroin addiction.

AIMS: The carotid body (CB) represents the prime site for detecting and responding to hypoxia. Since the role of heroin in respiratory depression with consequent hypoxia is known, the authors were able to investigate morphological and molecular modifications occurring in the CB of heroin addicted subjects compared to subjects who died because of trauma. METHODS AND RESULTS: CB sampled from six 27 year old subjects, slides were treated with Mallory Trichrome staining or used for immunohistochemical analysis to detect Neuroglobin (NGB), Hypoxia Inducible Factor-1 (HIF-1α), Vascular Endothelial Growth Factor (VEGF), Inducible Nitric Oxide Synthase (i-NOS), Bax and cleaved Caspase-3 proteins. Mallory Trichrome staining shows an increase in the connective tissue in heroin subjects compared to controls and a parallel reduction in parenchymal area. Immunohistochemical analyses in heroin subjects found a decrease in NGB and an increase in HIF-1α and VEGF compared to controls; i-NOS expression was not statistically significant. Bax and cleaved caspase-3 were positive only in the heroin subjects. CONCLUSIONS: These results could confirm the typical hypoxic condition occurring in heroin addicts. Since NGB may function as a reactive oxygen or nitrogen species scavenger and as apoptotic cell death protector, the decrease in its expression may suggest a key role of this globin in human CB impairment due to heroin addiction.

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.

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.

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.

Fluoxetine may worsen hyperoxia-induced lung damage in neonatal rats.

Fluoxetine shows controversial lung effects as it prevents pulmonary hypertension in adult rats but exposure during gestation causes pulmonary hypertension in neonatal rats. In the present study, we tested the null hypothesis that the antidepressant drug fluoxetine does not modify the development of bronchopulmonary dysplasia (BPD) in neonatal rats. Experimental categories included I: room air (controls) with daily injection of saline; II: room air with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks; III: 60% oxygen with daily injection of saline; and IV: 60% oxygen with daily injection of 10 mg/kg fluoxetine, i.p., during two weeks. Hyperoxia resulted in significant reduction in alveolar density and an increase in pulmonary endocrine cells, as well as increases in muscle layer areas of bronchi and arteries. Fluoxetine treatment generated a further increase in muscularisation and did not significantly modify the hyperoxia-induced reductions in alveolar density and increases in the endocrine cells. In hyperoxia, Real-Time PCR showed a lower pulmonary expression of vascular endothelial growth factor (VEGF) with no significant changes in the expression of matrix metalloproteinases (MMP) 2 and 12. Fluoxetine did not affect VEGF or MMP-2 expression but it significantly increased MMP-12 mRNA in both normoxic and hyperoxic groups. Zymographic analysis of MMP-2 activity in bronchoalveolar fluid showed a significantly reduced MMP-2 activity in hyperoxia, while fluoxetine treatment restored MMP-2 activity to levels comparable with the normoxic group. In conclusion, our data show that fluoxetine may worsen bronchial and arterial muscularisation during development of BPD and may up-regulate MMP expression or activity.

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.