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Giant cell tumor of bone (GCTB) is a locally aggressive primary bone neoplasm composed by tumoral stromal cells (SCs) and a reactive component that consists of monocytic/histiocytic cells that give rise by fusion to osteoclast-like multinucleated cells. Recently, specific Histone 3.3 mutations have been demonstrated in SCs of GCTB. Many of the pathways related to bone proliferation and regulation depend on the primary cilium, a microtubule-based organelle that protrudes outside the cell and acts as a sensorial antenna. In the present work, we aimed to study the presence and role of primary cilia in GCTB. Ultrastructural, immunohistochemical, and immunofluorescence studies were performed in order to demonstrate, for the first time, that the primary cilium is located in spindle-shaped SCs of GCTB. Moreover, we showed Hedgehog (Hh) signaling pathway activation in these cells. Hence, primary cilia may play a relevant role in GCTB tumorogenesis through Hh signaling activation in SCs. RESEARCH HIGHLIGHTS: Transmission electron microscopy allows describing and differentiating cellular subpopulations in giant cell tumor of bone (GCTB). The primary cilium is present in some tumoral stromal cells of GCTB. Hedgehog signalling is activated in these cells.

Gastrointestinal stromal tumour (GIST) is a mesenchymal cancer which derives from interstitial cells of Cajal. To determine whether a relationship between Hedgehog (Hh) signalling pathway and primary cilia exists in GIST tumours is intended here. Immunohistochemical, immunofluorescence and ultrastructural techniques were performed in this study. We show that GIST cells present primary cilia (an antenna-like structure based on microtubules). But, moreover, we prove Hedgehog signalling pathway activation in these tumours (a pathway related with tumoural features such as proliferation, migration or stemness) and we show for the first time that this signalling pathway activation in GIST is mediated by primary cilia, likely in a paracrine way. Thus, primary cilia and Hedgehog signalling would be fundamental in tumoural microenvironment control of GIST cells for their maintenance, differentiation and proliferation.

Irreversible electroporation (IRE) has gained attention as a new non-thermal therapy for ablation with important benefits in terms of homogeneous treatment and fast recovery. In this study, a new concept of high voltage generator is used, enabling irreversible electroporation treatment in large tissue volume using parallel plates. Unlike currently available generators, the proposed versatile structure enables delivering high-voltage high-current pulses. To obtain homogeneous results, 3-cm parallel-plates electrodes have also been designed and implemented. IRE ablation was performed on six female pigs at 2000 V/cm electric field, and the results were analysed after sacrifice three hours, three days and seven days after ablation. Histopathological and ultrastructural studies, including transmission and scanning electron microscopy, were carried out. The developed high-voltage generator has proved to be effective for homogeneous IRE treatment using parallel plates. The destruction of the membrane of the hepatocytes and the alterations of the membranes of the cellular organelles seem incompatible with cell death by apoptosis. Although endothelial cells also die with electroporation, the maintenance of vascular scaffold allows repairing processes to begin from the third day after IRE as long as the blood flow has not been interrupted. This study has opened new direction for IRE using high performance generators and highlighted the importance of taking into account ultrastructural changes after IRE by using electron microscopy analysis. This work was partly supported by the Spanish MINECO under Project TEC2016-78358-R and by the DGA-FSE. Authors would like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza.

Dedifferentiation is a loss of phenotypic specialization that converts differentiated cells into adult stem cells in order to proliferate and differentiate into replacement tissue. This occurs in several tissues from various organs, such as smooth muscle cells (SMCs) of the mammalian gastrointestinal tract. The aim of this study was to describe ultrastructural and immunohistochemical changes in SMCs which could be compatible with a dedifferentiation process in human and rabbit intestinal muscles. Ultrastructural study and immunohistochemical staining (SMemb and MyoD) on human and rabbit duodenum tissue sections were performed. In both species, this dedifferentiation process is characterized by a loss of intercellular junctions, increased intercellular spaces, cytoskeletal disorganization, perinuclear accumulation of large vacuoles that tend to fuse, rupture of the vacuole membrane and release of cytoplasmic fragments. Dedifferentiated cells show the characteristic phenotype of a mesenchymal cell with scarce perinuclear cytoplasm, long cytoplasmic prolongations and finely distributed granular chromatin in the nucleus. These morphological changes are accompanied by a modulation to a less mature phenotype showing immunoreactivity for the embryonic form of the myosin heavy chain and for the myogenic regulatory factor MyoD. We suggest that SMC dedifferentiation includes the elimination of the contractile apparatus, the activation of the nucleus and the re-expression of embryonic markers. We described an ultrastructural dedifferentiation process possible in intestinal SMCs. This dedifferentiation process seems to play a key role in the homeostasis of the intestinal muscle.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O2, a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O2) and hypoxia (5% O2). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Extracellular vesicles (EVs) have emerged as an intercellular communication mediator in cancer. They seem to be involved in tumor processes by means of transformation of surrounding cells previous to metastasis by transferring miRNAs and oncogenic proteins. It is known that EVs, depending on their source, can be exosomes or ectosomes. Although the first type constitutes a specific population formed from the endosomal system, via multivesicular bodies, the ectosome biogenesis is not yet well known. In this study, we report a new type of EVs which has been termed spheresomes. While exosomes come from multivesicular bodies and ectosomes from direct budding of plasma membrane, spheresomes present a new mechanism of shedding from a spherical membrane structure which we have named multivesicular spheres. These EVs are first described in gastrointestinal stromal tumor cells in the present study. But moreover, these new membrane spherical structures appear not only next to tumoral cells but also different distances from them. Since some other authors have evidenced oncogenic KIT-containing EVs, it is also suggested here that surrounding cells uptake of these first described EVs, GIST-derived spheresomes, could induce tumor invasiveness. That is why the prevention of signaling processes developed by these new EVs may represent an alternative approach for GIST treatment.

Background Electrical bioimpedance (BI) has been used to indirectly measure steatosis. This method has not yet been established in the clinics thus experimental studies are needed in big animals. We assessed BI to measure liver steatosis in porcine animals. Methods Twelve large-white × Landrace pigs weighing 35 kg were allocated to a study ( n = 9) and a control group ( n = 3). A special diet was used to promote steatosis among the study group: methionine deficient and choline-restricted diet that contains supplements of cholesterol, collate and excess of saturated fat. Control group animals were fed a normal diet. A new tetrapolar electrode model was used for BI measurement, which were performed during open laparotomy by inserting a probe into one of the lobes. Measurements were done in the third and fourth segments of the pig liver, placing the probe either on the surface or inserted into the parenchyma of the liver. Open biopsies were obtained at the end of the measurements. Histological samples were processed and stained with hematoxylin-eosin to estimate macrosteatosis. Pearson correlation coefficient between BI and percentage steatosis were calculated at different frequencies. Results After 4 months of the special diet all the animals in the study group developed steatosis (90% to 20%), whereas none of the control group was affected. Pearson correlation coefficients between BI and percentage of steatosis were significant (0.877–0.878) with the best correlations obtained with a probe placed on the fourth segment of the liver surface and the best frequency to perform the measurements being 50 and 75 kHz. Conclusions BI is an accurate, fast method for steatosis measurements, that is easier and cheaper than either open or needle biopsy.

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells that could potentially be used to repair injured cartilage in diseases such as osteoarthritis (OA). In this study we used bone marrow, adipose tissue from articular and subcutaneous locations, and synovial fluid samples from 18 patients with knee OA to find a suitable alternative source for the isolation of MSCs with high chondrogenic potential. MSCs from all tissues analysed had a fibroblastic morphology, but their rates of proliferation varied. Subcutaneous fat-derived MSCs proliferated faster than bone marrow- and Hoffa’s fat pad-derived MSCs, while synovial fluid-derived MSCs grew more slowly. CD36 and CD54 expression was similar across all groups of MSCs with several minor differences. High expression of these surface markers in subcutaneous fat-derived MSCs was correlated with poor differentiation into hyaline cartilage. Synovial fluid-derived MSCs presented a relatively small chondrogenic differentiation capacity while Hoffa’s fat pad-derived MSCs had strong chondrogenic potential. In conclusion, MSCs from elderly patients with OA may still display significant chondrogenic potential, depending on their origin.

This study analyzed the phenotype and the chondrogenic differentiation of bone marrow-derived MSCs from old patients undergoing knee osteoarthritis or femoral fracture surgery. Twenty patients (12 females), with a mean age of 77.35±8.76 years, were studied. Ten patients suffered of knee osteoarthritis (OA) pathology and underwent surgery for arthroplasty, and the other 10 patients suffered femoral fracture. A comparative study of bone marrow-derived cultured human MSCs was carried out, and the main morphological parameters, proliferative activity and expression of surface markers were characterized. Bone marrow was obtained from the femur in all cases. The χ2-test, Mann-Whitney U-test, correlation coefficient and the Spearman test were applied. Bone marrow MSCs from old patients were able to differentiate into chondrocytic lineages. Proliferation and flow cytometry data showed no difference associated to the gender. No significant differences between the knee arthroplasty group or the femoral fracture group were found, except for higher CD49d % in MSC from fracture, and higher CD49f % in MSC from knee OA patients at passage one. MSCs from old patients suffering knee OA can be differentiated into chondrocytic lineages, and these present no differences with MSCs from femoral fracture patients.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue-derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species-related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay.

Purpose The aim of the present article was to study the influence of platelets and different time activation on cartilage growth in articular defects in the rabbit knee. Methods Twelve male New Zealand rabbits (12 weeks) were divided in two groups. Under general anaesthesia, a 4 mm diameter and 2 mm deep defect was performed in medial condyles in both knees. The right knee defect was filled with platelet concentrate 5 min after being activated with ClCa in group A, and 2 min afterwards in group B. Platelets were obtained by centrifuging 10 ml arterial blood from the rabbit prior to the surgical procedure. The left knee defect was not filled. Rabbits were sacrificed 6 weeks after surgery. Macroscopic and microscopic studies were performed. Results In group A, hyaline cartilage was observed in the right knee defect at the end of the experiment in five rabbits. None of the defects of the left knees showed hyaline cartilage growth. In group B, hyaline cartilage was observed in the right knee defect in only one rabbit. Nevertheless, in group B, all rabbits presented better chondral cellularity and regeneration and lower fibrosis in defects treated with platelets than in non-treated ones. Conclusions This technique for articular defect reconstruction with platelets is simple and easy, and has shown satisfactory results in our study. Platelets may be useful as an autologous source of multiple growth factors for articular defect reconstruction. Nevertheless, this is a preliminary study and further research is required.

Locomotor system disorders in equine species, such as tendon lesions, osteoarthritis, or ligament injuries, are some of the most frequent causes of dramatic reduction in horse performance. Traditional therapies are aimed at the inflammatory process and pain, but they do not regenerate normal tendon or ligament matrix and do not reduce re-injured rates. Mesenchymal stem cells have started to use as therapeutic option to repair these injured tissues. Most studies have focused on their isolation, in vitro culture and pheno-typing. However, mesenchymal stem cell ultrastructure has been disregarded in the last years. We investigate the ultrastructural characteristics of these cells once differentiated into chondrocytes. Ultrastructural analysis was conducted on suspension cultures of differentiated chondrocytes from bone marrow mesenchymal stem cells by means of transmission electron microscopy. The morphologic characteristics of these cells, their ability to produce the extracellular matrix, and the presence of a single cilium could be indicative of the mesenchymal cells differentiation into chondrocyte phenotype. This study provides essential data to evaluate the degree of suitable phenotypic stability, for these cells can be used with repair purposes. (C) 2016 Elsevier Inc. All rights reserved.

Bipolar radiofrequency (RF) ablation, especially with perfusion of saline, has been shown to increase volume over monopolar conventional methods. The aims of this study are to study whether this method is linked to too flattened thermal lesions and premature rise of impedance and to elucidate some safety concerns. Eighteen RF ablations were performed using a 1.8-mm-diameter bipolar applicator in the liver of nine healthy pigs through laparotomy with or without temporary vascular occlusion [the Pringle maneuver (PGM)]: group A (n=9), without PGM; group B (n=9), with PGM. Hypertonic saline solutions (3% and 20 %) were injected through the applicator at a rate of 400 ml/h during the procedure. The pigs were followed up and they were euthanased on the 15th day. Impedance, current, power output, energy output, temperatures, diameters of thermal lesion, volume, sphericity ratio of thermal lesion were correlated among groups. Impedance at the end of the procedure (50.00 Omega+/-28.39 and 52.88 Omega+/-26.77, for groups A and B, respectively) was very similar to the starting impedance (50 Omega). In a median of 1 (range, 0-6) time per RF ablation procedure a reduction of 30 W from the selected power supply was observed during the RF ablation procedure linked to a slight increase of impedance. Volume and short diameter of thermal lesion were 21.28 cm3+/-11.78 and 2.85 cm+/-0.87 for group A, 87.51 cm3+/-25.20 and 4.31 cm+/-0.65 for group B. Continuous thermal between both electrodes were described with a global sphericity ratio of 1.91. One major complication (thermal injury to the stomach) was encountered in a case of cross-sectional necrosis of the targeted liver and attributed to heat diffusion after the procedure. This method has been shown to determine: (1) the relative control of impedance during the procedure; (2) ovoid and relatively large thermal lesions with less dependence upon closest vessels.

To evaluate whether a bipolar saline-enhanced radiofrequency (RF) ablation system embedded in one needle is able to consistently produce homogeneous and predictable areas of coagulation necrosis with or without the Pringle maneuver of vascular inflow occlusion.RF ablation (480 kHz) of the liver was performed in 24 healthy pigs by means of laparotomy: group A (n = 5), 4-cm distance between electrodes 1 and 2; group B (n = 7), 4-cm distance and the Pringle maneuver; group C (n = 5), 2-cm distance; and group D (n = 7), 2-cm distance with the Pringle maneuver. Twenty percent NaCl solution was infused continuously at a rate of 100 mL/h via each electrode during the procedure. The pigs were followed up, and they were euthanized on the 7th day. Livers were removed for histologic assessment. Time, impedance, current, power output, specific voltage of the contacts, energy output, temperatures in the liver, volume of the lesion, and energy delivered per lesion volume were determined and compared among groups. Predictability of lesion volume was evaluated with the coefficient of variability. Mean values of the variables were compared among the groups by means of one-way analysis of variance or Kruskall-Wallis test.Impedance at the end of the RF ablation procedure was almost twofold lower than the corresponding initial value in all groups. In Pringle groups B and D, regular ellipsoids of coagulation necrosis were created (mean lesion volume, 149.50 cm3 +/- 34.26 and 69.43 cm3 +/- 15.48, respectively). In non-Pringle groups A and C, the shape of coagulation necrosis was influenced by the vessels encountered, and mean lesion size was lower than that in the Pringle groups (P.01). The coefficient of variability of lesion size was lower in the Pringle groups (23% and 22%, respectively) than that in the non-Pringle groups (75% and 30%, respectively).The bipolar saline-enhanced RF ablation method produces homogeneous and predictable areas of coagulation necrosis between two electrodes, regardless of the distance between them, preferably with vascular inflow occlusion.

The authors' purpose was to create larger and more regular liver lesions in vitro by testing a new hyperthermia approach that uses a bipolar saline-enhanced electrode for radiofrequency (RF) in the audible spectrum and a greater power supply.The authors' hyperthermia approach (group A, n = 23) was used in excised porcine livers, and the results were compared with those of a previously described monopolar saline-enhanced electrode procedure (group B, n = 23). In each set of experiments, RF in the audible spectrum current (50 Hz) was provided for 15 minutes with a similar ablation protocol. Electrical variables (impedance, current, voltage, power, and energy), temperatures in the lesions, volume size, regularity ratio of the lesion, and microscopic findings were measured.In group A, the mean volume size and the mean regularity ratio values were 144.8 cm3 +/- 59.8 and 0.78 +/- 0.1, respectively. In group B, the mean volume size and regularity ratio values were 62.1 cm3 +/- 36.4 and 0.62 +/- 0.1, respectively. The values in group B were thus significantly lower than those in group A (P.01). The lesions in group A were also more homogeneous. No significant differences were found in electrical variables.The new bipolar saline-enhanced electrode produced larger, more regular, and more homogeneous lesions ex vivo than the previously used monopolar saline-enhanced electrode method. Using a greater power supply increased the amount of coagulative necrosis.

In order to deepen our knowledge of the different components of the chicken intestinal nerve of Remak (I.N.R.), we have studied it by means of histochemical, immunohistochemical and electron microscopy techniques to distinguish the different neurotransmitters. We have found cholinergic cell bodies, as well as acetylcholinesterase (AChE) positive neuronal fibers, forming part of the web that constitutes the I.N.R. in its caudal portion, with a higher density of neuronal bodies in the ganglia. We also observed catecholaminergic neuronal bodies and fibers, located fundamentally in the periphery of the nerve, and a low density of catecholaminergic cell bodies. With respect to the vasoactive intestinal peptide (VIP) and substance P (SP) positive peptidergic innervation, we found more abundant neuronal bodies positive for the V.I.P. than for S.P. Electron microscopy corroborated the results observed under the optic microscope, showing the various types of vesicles containing different neurotransmitters.

We study the esophagus of Podarcis hispanica through different methods to clarify the structure and affinities of its wall innervation. The acetylcholinesterase method reveals cholinesterase activity in two submucosal nervous plexuses, with an increasing degree of structural complexity in the reptilian esophagus, compared with amphibians. Noradrenergic innervation, detected through fluorescence induced by formol, widely spreads its network in both the myenteric and submucosal plexuses (around the blood vessels in the external submucosal plexus, and to the glandular lamina propria in the inner submucosal plexus). Immunohistochemistry for vasoactive intestinal peptide shows a widespread innervation, with neurons clustered in ganglia and also scattered through the VIPergic network, only at the myenteric plexus. Immunohistochemistry for substance P shows a rich innervation along the entire wall of the esophagus, more concentrated in its caudal region, around the blood vessels. Electron microscopy shows the enteric neuronal ultrastructure and its relationship with the esophagus wall.

Studies in vitro have permitted the identification of enteric neural progenitor cells. Now the question arises as to where these progenitor cells are located in vivo. The purpose of this paper is to identify possible candidate cells by means of transmission electron microscopy (TEM). We have located three interstitial cellular types around the rat duodenum myenteric plexus. Type I cells have been identified as Interstitial Cells of Cajal (ICCs). These cells present well defined ultrastructural characteristics, including the triple connexion IC- nervous trunk- blood vessels. Type II cells show characteristics of immature cells, emphasizing the presence of a single cilium with the structure (9+0). To analyse this nanostructure, we have elaborated a reconstruction on ultrathin sections. The two previously described cellular types could be considered to be different functional states of the same cell. Type III cells present ultrastructural characteristics of fibroblast-like cells. This study suggests that Type II cells could be a source of neural progenitor cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue-derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species-related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay.

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duo-denum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce per-inuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular con-tacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium show-ing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.

The aim of any device designed for liver resection is to allow blood saving and quick resections. This may be optimized using a minimally invasive approach. A radiofrequency-assisted device is described that combines a cooled blunt-tip electrode with a sharp blade on one side in an in vivo preliminary study using hand-assisted laparoscopy to perform partial hepatectomies.Eight partial hepatectomies were performed on pigs with hand-assisted laparoscopy using the radiofrequency-assisted device as the only method for transection and hemostasis. The main outcome measures were transection time, blood loss, transection area, transection speed, blood loss per transection area, and tissue coagulation depth. The risk for biliary leak also was assessed using the methylene blue test.The transection time was 13 +/- 7 min for a mean transected area of 34 +/- 11 cm(2). The mean total blood loss was 26 +/- 34 ml. The mean transection speed was 3 +/- 1 cm(2)/min, and the blood loss per transection area was 1 +/- 1 ml/cm(2). Abdominal examination showed no complications in nearby organs. One biliary leak was identified in one case using the methylene blue test. The transection surface was 34 +/- 11 cm(2), and the mean tissue coagulation depth was 9 +/- 2 mm. The inviability of the coagulated surface was assessed by adenine dinucleotide (NADH) staining.The radiofrequency-assisted device has shown with a laparoscopic approach that it can perform liver resections faster and with less blood loss using a single device in a minimally invasive manner without vascular control than other commercial devices. The results show no significant differences with the same device used in an open procedure.

Background Radiofrequency ablation (RFA) of tumors by means of internally cooled electrodes (ICE) combined with interstitial infusion of saline may improve clinical results. To date, infusion has been conducted through outlets placed on the surface of the cooled electrode. However, the effect of infusion at a distance from the electrode surface is unknown. Our aim was to assess the effect of perfusion distance (PD) on the coagulation geometry and deposited power during RFA using ICE. Methods Experiments were performed on excised bovine livers. Perfusion distance (PD) was defined as the shortest distance between the infusion outlet and the surface of the ICE. We considered three values of PD: 0, 2 and 4 mm. Two sets of experiments were considered: 1) 15 ablations of 10 minutes (n ≥ 4 for each PD), in order to evaluate the effect of PD on volume and diameters of coagulation; and 2) 20 additional ablations of 20 minutes. The effect of PD on deposited power and relative frequency of uncontrolled impedance rises (roll-off) was evaluated using the results from the two sets of experiments (n ≥ 7 for each PD). Comparisons between PD were performed by analysis of variance or Kruskal-Wallis test. Additionally, non-linear regression models were performed to elucidate the best PD in terms of coagulation volume and diameter, and the occurrence of uncontrolled impedance rises. Results The best-fit least square functions were always obtained with quadratic curves where volume and diameters of coagulation were maximum for a PD of 2 mm. A thirty per cent increase in volume coagulation was observed for this PD value compared to other values (P < 0.05). Likewise, the short coagulation diameter was nearly twenty five per cent larger for a 2 mm PD than for 0 mm. Regarding deposited power, the best-fit least square function was obtained by a quadratic curve with a 2 mm PD peak. This matched well with the higher relative frequency of uncontrolled impedance rises for PD of 0 and 4 mm. Conclusion Saline perfusion at around 2 mm from the electrode surface while using an ICE in RFA improves deposition of energy and enlarges coagulation volume.

The aim of the present article was to study the influence of platelets and different time activation on cartilage growth in articular defects in the rabbit knee.Twelve male New Zealand rabbits (12 weeks) were divided in two groups. Under general anaesthesia, a 4 mm diameter and 2 mm deep defect was performed in medial condyles in both knees. The right knee defect was filled with platelet concentrate 5 min after being activated with ClCa in group A, and 2 min afterwards in group B. Platelets were obtained by centrifuging 10 ml arterial blood from the rabbit prior to the surgical procedure. The left knee defect was not filled. Rabbits were sacrificed 6 weeks after surgery. Macroscopic and microscopic studies were performed.In group A, hyaline cartilage was observed in the right knee defect at the end of the experiment in five rabbits. None of the defects of the left knees showed hyaline cartilage growth. In group B, hyaline cartilage was observed in the right knee defect in only one rabbit. Nevertheless, in group B, all rabbits presented better chondral cellularity and regeneration and lower fibrosis in defects treated with platelets than in non-treated ones.This technique for articular defect reconstruction with platelets is simple and easy, and has shown satisfactory results in our study. Platelets may be useful as an autologous source of multiple growth factors for articular defect reconstruction. Nevertheless, this is a preliminary study and further research is required.

The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.

The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.

Background Efficient and safe liver parenchymal transection is dependent on the ability to address both parenchymal division and hemostasis simultaneously. In this article we describe and compare with a saline-linked instrument a new radiofrequency (RF)-assisted device specifically designed for tissue thermocoagulation and division of the liver used on an in vivo pig liver model. Methods In total, 20 partial hepatectomies were performed on pigs through laparotomy. Two groups were studied: group A ( n =8) with hepatectomy performed using only the proposed RF-assisted device and group B ( n =8) with hepatectomy performed using only a saline-linked device. Main outcome measures were: transection time, blood loss during transection, transection area, transection speed and blood loss per transection area. Secondary measures were: risk of biliary leakage, tissue coagulation depth and the need for hemostatic stitches. Tissue viability was evaluated in selected samples by staining of tissue NADH. Results In group A both blood loss and blood loss per transection area were lower ( p =0.001) than in group B (70±74ml and 2±2ml/cm 2 vs. 527±273ml and 13±6ml/cm 2 , for groups A and B, respectively). An increase in mean transection speed when using the proposed device over the saline-linked device group was also demonstrated (3±0 and 2±1cm 2 /min for group A and B, respectively) ( p =0.002). Tissue coagulation depth was greater ( p =0.005) in group A than in group B (6±2mm and 3±1mm, for groups A and B, respectively). Neither macroscopic nor microscopic differences were encountered in transection surfaces between both groups. Conclusions The proposed RF-assisted device was shown to address parenchymal division and hemostasis simultaneously, with less blood loss and faster transection time than saline-linked technology in this experimental model.

[EN] Objective: Determine whether Radiofrequency ablation (RFA) of the pancreas and subsequent transection of the main pancreatic duct may avoid the risk of both necrotizing pancreatitis and postoperative pancreatic fistula (POPF) formation. Methods: Thirty-two rats were subjected to RFA and section of the pancreas over their portal vein. Animals were killed at 3, 7, 15 and 21 days (Groups 0-3, respectively). Two additional control groups (sham operation and user manipulation only, respectively) of 15 days of postoperative period were considered. Postoperative complications, histological changes (including morphometric and immunohistochemical analysis) and incidence of POPF were evaluated. Results: A significant increase in serum amylase levels (p, This research has been supported by grant TEC2011-27133-C02-02 from the Spanish Government and the European Economic Community.

Sir .—Osteoid osteoma is a benign bone tumor that principally affects men in the second decade of life. The clinical manifestations include a characteristic pain with nocturnal exacerbations that recedes or disappears after taking aspirin. In addition, muscular atrophy in the affected extremity and functional impairment are noted. If the tumor is located in the vertebrae, scoliosis can result. The most frequent locations are the femur, tibia, vertebrae, and humerus. A preliminary diagnosis can be made clinically, but confirmation is made by the visualization of the pathognomonic bone lesion, which is the "nidus" surrounded by an area of sclerosis. After surgical treatment, pathological study can confirm the diagnosis by examination of biopsy specimens. We present a series of patients with osteoid osteoma in whom computed tomography assisted in diagnosis of the lesions. Patient Reports .—Six children ranging in age from 3 years 5 months to 12 years 6 months

1. The location, distribution and morphological characteristics of the pigment cells found in the frog gut are described. 2. The pigment cells show long and large protoplasmic projections. At the ultrastructural level, the nucleus is elongated with prominent nucleolus and dense marginal chromatin. The cytoplasm is full with pigment granules (2500-7500 A) and typical premelanosome structures have been observed. 3. The pigment cells number is higher in the esophagus and large intestine than in the stomach or small intestine and the pigment cells are always located in close contact with blood vessels and nervous structures (ganglia and fibres). 4. We have observed that the pigment content depends upon seasonal variations, increasing during the cold months. 5. We have demonstrated by histological methods that the cells pigment content is melanin. 6. According to their morphological and tinctorial characteristics the anuran gut melanin storing cells are similar to the skin epidermal melanocytes.

3‐Hydroxy‐3‐methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3‐hydroxy‐3‐methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG‐CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients. info:eu-repo/semantics/acceptedVersion

Background Efficient and safe transection of biological tissue in liver surgery is strongly dependent on the ability to address both parenchymal division and hemostasis simultaneously. In addition to the conventional clamp crushing or finger fracture methods other techniques based on radiofrequency (RF) currents have been extensively employed to reduce intraoperative blood loss. In this paper we present our broad research plan for a new RF-assisted device for bloodless, rapid resection of the liver. Methods Our research plan includes computer modeling and in vivo studies. Computer modeling was based on the Finite Element Method (FEM) and allowed us to estimate the distribution of electrical power deposited in the tissue, along with assessing the effect of the characteristics of the device on the temperature profiles. Studies based on in vivo pig liver models provided a comparison of the performance of the new device with other techniques (saline-linked technology) currently employed in clinical practice. Finally, the plan includes a pilot clinical trial, in which both the new device and the accessory equipment are seen to comply with all safety requirements. Results The FEM results showed a high electrical gradient around the tip of the blade, responsible for the maximal increase of temperature at that point, where temperature reached 100°C in only 3.85 s. Other hot points with lower temperatures were located at the proximal edge of the device. Additional simulations with an electrically insulated blade produced more uniform and larger lesions (assessed as the 55°C isotherm) than the electrically conducting blade. The in vivo study, in turn, showed greater transection speed (3 ± 0 and 3 ± 1 cm2/min for the new device in the open and laparoscopic approaches respectively) and also lower blood loss (70 ± 74 and 26 ± 34 mL) during transection of the liver, as compared to saline-linked technology (2 ± 1 cm2/min with P = 0.002, and 527 ± 273 mL with P = 0.001). Conclusion A new RF-assisted device for bloodless, rapid liver resection was designed, built and tested. The results demonstrate the potential advantages of this device over others currently employed.