Localization of telocytes in rabbits testis: Histological and immunohistochemical approach.

Telocyte (TC) is an interesting unique interstitial cell demonstrated in many human and animal tissues and organs. This study verified, for the first time, the pattern of TC distribution in the testicular tissue of New Zealand White rabbits using histological, immunohistochemical, and electron microscopic tools. Rabbit testicular tissue samples were obtained from three pairs of adult healthy New Zealand white rabbit by surgical procedures. The testicular tissues were stained with hematoxyline-eosin, Crossmon's trichrome and Periodic acid Schiff. The immunohistochemistry was performed using three different antibodies CD34, CD117, and vimentin. The testes were examined by scanning and transmission electron microscopy. Histologically, TCs formed a sheath surrounding the seminiferous tubules. Other TCs were located in the interstitial tissue of the rabbit testis. Immunohistochemically, TCs reacted strongly with CD34, CD117, and vimentin. Scanning electron microscopic findings clearly elucidated the spreading pattern of TCs and their cytoplasmic processes with the interstitial tissue including blood vessels. Both homocellular and heterocellular junctions were demonstrated by transmission electron microscope. On the basis of TCs distribution and connections, the before mentioned data suggested that, TCs may play a potential role in maintaining the testicular construction and regulation. A future work is needed to clarify the actual role played by TCs in monitoring testicular fertility. RESEARCH HIGHLIGHTS: Telocyte (TC) is a unique cell demonstrated in human and animal tissues. TCs formed a sheath surrounding the seminiferous tubules in rabbits and may be located in interstitial tissue. Immunohistochemically, TCs reacted strongly with CD34 and CD117.

Morphology of the interstitial tissue of active and resting testis of the Guinea fowl / Morfología del tejido intersticial del testículo activo y en reposo de la gallina de Guinea

SUMMARY: The morphology of the interstitial tissue of sexually active and resting testis of the guinea fowl were studied. Six adult health birds of active and resting phases of reproductive cycle were used for this study. The interstitial tissue consisted of loose connective tissue, interstitial cells (Leydig cells), few connective cells, blood vessels and adrenergic nerve fibres in the present study in both active and resting testes. The interstitial tissue was compact in sexually active testis whereas, the volume of the tissue was found to be increased in resting testis. The loose connective tissue of the interstitial tissue composed of mainly of collagen fibres and few reticular fibres whereas elastic fibres were absent in both groups studied. The interstitial cells appeared in clusters of a few cells and were relatively less in the active testis than the resting testis. The interstitial cells were pale staining or polygonal cells with euchromatic nuclei with few large lipid droplets in the active testis whereas the cells were flat and highly heterochromatic with numerous small lipid droplets in resting testis. Few macrophages were found only in resting testis. Interstitial cells showed negative reaction to alkaline, acid phosphatases and PAS in both groups studied but positive for lipids. The interstitial tissue was well vascularised with centrally located blood vessels in the active testis whereas the blood vessels were small and inconspicuous in the resting testis. The lymphatic vessels were not identified in both groups studied.

RESUMEN: Se estudió la morfología del tejido conectivo intersticial en testículos sexualmente activos y en reposo de la gallina de Guinea (Numida meleagris). Se utilizaron seis gallinas de Guinea machos adultos sanos, en fase activa y de reposo del ciclo reproductivo. El tejido conectivo intersticial estaba formado por tejido conectivo laxo, células endocrinas intersticiales, pocas células conectivas, vasos sanguíneos y fibras nerviosas adrenérgicas, tanto en testículos activos como en reposo. El espacio intertubular en los testículos sexualmente activos era menor en comparación a los del testículos en reposo. El tejido conectivo laxo estaba compuesto principalmente de fibras colágenas y en menor cantidad de fibras reticulares. Las fibras elásticas estaban ausentes en ambos grupos. Las células endocrinas intersticiales se organizaban en racimos de pocas células y se observaban con menor frecuencia en los testículos sexualmente activos. Las células endocrinas intersticiales de los testículos activos presentaban forma poligonal, citoplasma levemente eosinofílico con algunas gotas lipídicas de gran tamaño en su interior y nucleos redondos con cromatina laxa. Las células intersticiales de los testículos en reposo eran planas y altamente heterocromáticas, con numerosas gotas lipídicas pequeñas en su citoplasma. Las células intersticiales mostraron una reacción negativa a las fosfatasas ácidas, alcalinas y PAS en ambos grupos, Sin embargo presentaron reacción positivas para los lípidos. El tejido conectivo intersticial estaba bien vascularizado con vasos sanguíneos situados centralmente en el testículo activo y vasos sanguíneos pequeños y discretos en el testículo en reposo. Los vasos linfáticos no fueron identificados en los dos grupos estudiados.Los macrófagos fueron observados solo en los testículos en reposo, aunque en escasa cantidad.

Lipid-body containing interstitial cells (lipofibroblasts) in the lungs of various mouse strains.

Pulmonary alveolar septa are thought to contain at least two types of fibroblasts that are termed myofibroblasts and lipofibroblasts based on their morphological characteristics. Lipofibroblasts possess cytoplasmic lipid inclusions (lipid bodies or droplets) and are involved in several important functions, such as surfactant synthesis, development, vitamin A storage and presumably regeneration. As vitamin A was shown to reduce pulmonary emphysema in several but not all mouse and rat strains, we hypothesized that these strain differences might be explained by a differential occurrence of lipofibroblasts and their lipid bodies in various mouse strains. Therefore, mouse lungs of six strains (NMRI, BALB/c, C3H/HeJ, C57BL/6J, C57BL/6N and FVB/N) were investigated by light and electron microscopic stereology to quantify the amount of lipid bodies and the composition of alveolar septa. Lipofibroblasts were observed qualitatively by transmission electron microscopy in every investigated mouse strain. The total volume and the volume-weighted mean volume of lipid bodies were similar in all mouse strains. The results on the composition of the interalveolar septa did not show major differences between the groups. The only mouse strain that differed significantly from the other strains was the NMRI strain because the lungs had a higher volume and consequently many of the morphological parameters were also larger than in the other groups. In conclusion, the present study showed that lipofibroblasts are a common cell type in the mouse lung across various strains. Therefore, the mere presence or absence of lipofibroblasts does not explain differences in the pulmonary regenerative potential among mouse strains.

New insights into the cellular makeup and progenitor potential of palatal connective tissues.

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco-gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de-epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de-epithelialised MSCs (deMSCs) and re-entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony-forming unit- fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA-DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA-, CD31-) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,-in the pericapillary regions and in remote regions of the lamina propria- and pericytes-surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision-making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.

Calcium Signaling in Interstitial Cells: Focus on Telocytes.

In this review, we describe the current knowledge on calcium signaling pathways in interstitial cells with a special focus on interstitial cells of Cajal (ICCs), interstitial Cajal-like cells (ICLCs), and telocytes. In detail, we present the generation of Ca2+ oscillations, the inositol triphosphate (IP3)/Ca2+ signaling pathway and modulation exerted by cytokines and vasoactive agents on calcium signaling in interstitial cells. We discuss the physiology and alterations of calcium signaling in interstitial cells, and in particular in telocytes. We describe the physiological contribution of calcium signaling in interstitial cells to the pacemaking activity (e.g., intestinal, urinary, uterine or vascular pacemaking activity) and to the reproductive function. We also present the pathological contribution of calcium signaling in interstitial cells to the aortic valve calcification or intestinal inflammation. Moreover, we summarize the current knowledge of the role played by calcium signaling in telocytes in the uterine, cardiac and urinary physiology, and also in various pathologies, including immune response, uterine and cardiac pathologies.

Mitochondrial Ca²âº handling is crucial for generation of rhythmical Ca²âº waves in vascular interstitial cells from rabbit portal vein.

Vasomotion is the rhythmical changes in vascular tone of various blood vessels. It was proposed that in rabbit portal vein (RPV) the spontaneous contractile activity is driven by vascular interstitial cells (VICs), since RPV VICs generate rhythmical changes in intracellular Ca(2+) concentration ([Ca(2+)]i) associated with membrane depolarisation in these cells. In this work, using confocal imaging in Fluo-3 loaded RPV VICs we studied if generation of rhythmical [Ca(2+)]i changes is affected when Ca(2+) handling by mitochondria is compromised. We also visualised mitochondria in VICs using Mito Tracker Green fluorescent dye. Our results showed that freshly dispersed RPV VICs generated rhythmical [Ca(2+)]i oscillations with a frequency of 0.2-0.01 Hz. Imaging of VICs stained with Mito Tracker Green revealed abundant mitochondria in these cells with a higher density of the organelles in sub-plasmalemmar region compared to the central region of the cell. Oligomycin, an ATP synthase inhibitor, did not affect the amplitude and frequency of rhythmical [Ca(2+)]i oscillations. In contrast, two uncoupling agents, carbonylcyanide-3-chlorophenylhydrazone (CCCP) and carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP) effectively abolished rhythmical [Ca(2+)]i changes with simultaneous increase in basal [Ca(2+)]i in RPV VICs. These data suggest that in RPV VICs mitochondrial Ca(2+) handling is important for the generation of rhythmical [Ca(2+)]i changes which underlie the spontaneous rhythmical contractile activity in this vessel.

Morphological evidence of telocytes in mice aorta.

BACKGROUND: Telocytes (TCs) are a novel type of interstitial cells, which have been recently described in a large variety of cavitary and noncavitary organs. TCs have small cell bodies, and remarkably thin, long, and moniliform prolongations called telopodes (Tps). Until now, TCs have been found in various loose connective tissues surrounding the arterioles, venules, and capillaries, but as a histological cellular component, whether TCs exist in large arteries remains unexplored. METHODS: TCs were identified by transmission electron microscope in the aortic arch of male C57BL/6 mice. RESULTS: TCs in aortic arch had small cell bodies (length: 6.06-13.02 µm; width: 1.05-4.25 µm) with characteristics of specific long (7.74-39.05 µm), thin, and moniliform Tps; TCs distributed in the whole connective tissue layer of tunica adventitia: TCs in the innermost layer of tunica adventitia, located at the juncture between media and adventitia, with their long axes oriented parallel to the outer elastic membrane; and TCs in outer layers of tunica adventitia, were embedded among transverse and longitudinal oriented collagen fibers, forming a highly complex three-dimensional meshwork. Moreover, desmosomes were observed, serving as pathways connecting neighboring Tps. In addition, vesicles shed from the surface of TCs into the extracellular matrix, participating in some biological processes. CONCLUSIONS: TCs in aorta arch are a newly recognized complement distinct from other interstitial cells in large arteries, such as fibroblasts. And further biologically functional correlations need to be elucidated.

Telocytes in human term placenta: morphology and phenotype.

In the last few years, a new cell type - interstitial Cajal-like cell (ICLC) - has been described in digestive and extra-digestive organs. The name has recently been changed to telocytes (TC) and their typical thin, long processes have been named telopodes (TP). To support the hypothesis that TC may also be present in human placenta and add to the information already available, we provide evidence on the ultrastructure, immunophenotype, distribution, and interactions with the surrounding stromal cells of TC in the villous core of human term placenta. We used phase-contrast microscopy, light microscopy of semithin sections, transmission electron microscopy, immunohistochemistry, and immunofluorescence of tissue sections or cell cultures, following a pre-established diagnostic algorithm. Transmission electron microscopy showed cells resembling TC, most (∼76%) having 2-3 very thin, longprocesses (tens to hundreds of micrometers), with an uneven calibre(≤0.5 µm thick) and typical branching pattern. The dilations of processes accommodate caveolae, endoplasmic reticulum cisternae, and mitochondria. These TC have close contacts with perivascular SMC in stem villi. In situ, similar cells are positive for c-kit, CD34, vimentin, caveolin-1, vascular endothelial growth factor (VEGF), and inducible nitric oxide synathase (iNOS). The c-kit-positive cells inconsistently co-express CD34, CD44, αSMA, S100, neuron-specific enolase, and nestin. Among cells with a morphologic TC profile in cell cultures, about 13% co-express c-kit, vimentin, and caveolin-1; 70% of the c-kit-positive cells co-express CD34 and 12% co-express iNOS or VEGF. In conclusion, this study confirms the presence of TC in human term placenta and provides their ultrastructural and immunophenotypic characterization.

The microvacuolar system: how connective tissue sliding works.

The term 'fascia' has been applied to a large number of very different tissues within the hand. These range from aligned ligamentous formations such as the longitudinal bands of the palmar fascia or Grayson's and Cleland's ligaments, to the loose packing tissues that surround all of the moving structures within the hand. In other parts of the body the terms 'superficial' and 'deep fascia' are often used but these have little application in the hand and fingers. Fascia can be divided into tissues that restrain motion, act as anchors for the skin, or provide lubrication and gliding. Whereas the deep fascia is preserved and easily characterized in anatomical dissection, the remaining fascial tissue is poorly described. Understanding its structure and dynamic anatomy may help improve outcomes after hand injury and disease. This review describes the sliding tissue of the hand or the 'microvacuolar system' and demonstrates how movement of tissues can occur with minimal distortion of the overlying skin while maintaining tissue continuity.

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

Epithelial and PGP9.5-immunoreactive cells of Malassez epithelium in the periodontal ligament of cats: a transmission electron microscopic study.

OBJECTIVE: The aim of the study was to investigate the ultrastructural features of Malassez epithelium (ME) containing protein gene product 9.5 (PGP9.5)-immunoreactive (IR) cells in the cat periodontal ligament (PDL). MATERIAL AND METHODS: Specimens from the teeth and tooth-supporting tissues of four mature cats of both sexes, 18 to 24 months of age, were used. The fixed jaws were decalcified in EDTA. Frozen sagittal sections 20 µm thick were immunostained for PGP9.5, and the ME, containing IR cells in the PDL, were evaluated under a transmission electron microscope. RESULTS: Several epithelial cells and PGP9.5 IR cells formed clusters and were enveloped by a basal lamina and separated from the surrounding connective tissue. A large nucleus and scanty cytoplasm were observed in most of the ME cells, which contained abundant keratin filaments and mitochondria. Caveolae-like structures and vesicles were found in the periphery of the ME. The small cytoplasmic processes of some of the epithelial cells extended toward the surrounding connective tissues. The cytoplasmic matrix of one type of cell comprising the ME showed immunoreactivity for anti-PGP9.5 antibody. The IR cell in the cell clusters was connected to adjacent epithelial cells and extended cytoplasmic processes toward the adjacent epithelial cells. The IR cell contained keratin filaments and abundant densely cored vesicles approximately 100-250 nm in diameter. CONCLUSIONS: The findings of the study suggest endocytotic capabilities of the epithelial cells and neuroendocrine functions of the IR cells. It is possible that the two different cell types react to extrinsic stimuli and interact with cells comprising the clusters and cords in the PDL. These ultrastructural evidences may imply functional heterogeneity of the ME in the PDL.

Structure and function of rat lymph nodes.

The lymph node comprises a critical crossroad for encounters between antigen presenting cells, antigens from lymph, and lymphocytes recruited into lymph nodes from the blood. The node consists of spaces lined with lymphatic endothelial cells and parenchyma. The former spaces can be divided into the subcapsular sinuses, lymphatic labyrinths in the deep cortex, intermediate sinuses, and medullary sinuses. The sponge-like framework of the node parenchyma is composed of collagen fibers invested with reticular cells. The parenchyma can be divided into the cortex, deep cortex, and medullary cord. Lymphocytes migrate from the node parenchyma into the lymphatic labyrinths in the deep cortex. Close to the labyrinths are high endothelial venules (HEVs), through which circulating lymphocytes enter the node parenchyma. HEVs strongly express Aquaporin-1, suggesting that HEVs are involved in the net absorption of water, but not protein, from lymph coming through afferent lymphatics. Many LYVE-1 positive sinus reticular cells (i.e., lymphatic endothelial cells) with attached macrophages form a network within the lumen of the medullary sinuses. Fluids and migrating cells arriving at the node preferentially flow through the subcapsular sinuses, intermediate sinuses, and medullary sinuses in this order. Fluids and migrating cells may also enter the cortex through gaps in the floor of the subcapsular sinuses.

A Comparative Histomorphometric Study of the Stomach of Rat (Rattus norvegicus), Bat (Eidolon helvum) and Pangolin (Manis tricuspis) in Relation to Diet / Estudio Comparativo Histomorfométrico del Estómago de Rata (Rattus norvegicus), Murciélago (Eidolon helvum) y Pangolin (Manis tricuspis) en Relación con la Dieta

This study verified the comparative histomorphometric adaptations in the stomach of rat, bat and pangolin in relation to diet. Ten rats, ten bats and ten pangolins of both sexes were used for this investigation. The animals were sacrificed after slight anesthesia under chloroform inhalation. The stomach were excised, fixed in 10 percent formol saline and processed for light microscopic study. Stained slides were also subjected to morphometric analysis at a magnification of 400x. The results revealed that the cellular diameter/ density of parietal and zymogenic cells are significantly different in the three mammals (p<0.05) with the exception of the diameter of the zymogenic cells in pangolin which was not statistically significant (p>0.05) when compared with that of rat. Also, histological analysis revealed slight differences in the pattern of organization and distribution of connective tissue fibers. All these observations were reflections of the different pattern the stomachs of the three mammals have adopted to cope with their respective diets.

En este estudio se verificaron las adaptaciones histomorfométricas comparativas en el estómago de ratas, murciélagos y pangolines en relación a la dieta. Se utilizaron para esta investigación 10 ejemplares de cada especie, de ambos sexos. Los animales fueron sacrificados después de anestesia bajo inhalación de cloroformo. Los estómagos fueron extirpados, fijados en formol al 10 por ciento de solución salina y procesados para su estudio microscópico de luz. Los cortes teñidos fueron también objeto de análisis morfométrico con un aumento de X 400. Los resultados revelaron que el diámetro/densidad celular de parietal y las células cimógenas son significativamente diferentes en los tres mamíferos (p <0,05), con la excepción del diámetro de la células cimógenas de pangolines que no era estadísticamente significativa (p> 0,05) en comparación con la de rata. Por otra parte, el análisis histológico reveló ligeras diferencias en las características de organización y distribución de las fibras del tejido conjuntivo. Todas estas observaciones son un reflejo del patrón de los diferentes estómagos de los tres mamíferos, que han adoptado para hacer frente a sus respectivas dietas.

The primary cilium of connective tissue cells: imaging by multiphoton microscopy.

Although the role of the primary cilium as a sensory organelle in epithelial cells has been elucidated significantly over the past decade, the function of primary cilia in connective tissue cells has been studied less extensively. Primary cilia have been implicated as mechanotransducers in connective tissues, but the mechanisms by which the cells sense loads and convert them to biochemical signals for tissue formation and adaptation are poorly understood. Before hypotheses regarding the function of the primary cilium in connective tissue cells can be tested, methods for quantitation of incidence as well as three-dimensional visualization of primary cilia with respect to the extracellular matrix (ECM) are needed. The objective of this study was to develop a rapid method for visualizing primary cilia in their native ECM in a wide range of connective tissues. Whole-mount immunohistochemical and multiphoton microscopy techniques were developed to simultaneously image primary cilia, cell nuclei, and collagen and their relationships to each other in situ. Axonemes of primary cilia projecting into the ECM were successfully visualized in thick sections of growth plate cartilage, tendon, ligament, meniscus, intervertebral disc, and perichondrium. These methodologies will allow analysis of the incidence and three-dimensional orientation of primary cilia and enable investigation of the role of primary cilia in normal and pathological growth and adaptation in a variety of musculoskeletal tissues.

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach.

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.

Histochemical and ultrastructural characteristics of an interstitial cell type different from ICC and resident in the muscle coat of human gut.

CD117 (or c-kit) is expressed by the interstitial cells of Cajal (ICC), which are located within the gastrointestinal (GI) muscle coat and directly involved in its motility. CD34 is expressed by several cell types some of which have features and location resembling the ICC; however, a sure identification of these cells is still lacking. In order to establish whether the CD34-positive cells of the human GI tract are to be considered as ICC subpopulation or a novel independent cell type, and to hypothesize their nature and role, we verified CD34 and CD117 receptor expression under light and fluorescence microscope and performed a routine and a CD34-immuno-electron microscopy. CD34-positive cells were seen in the entire human GI tract. In the muscularis propria, shared morphologies similar to the c-kit-positive cells, in the submucosa, resembled fibroblasts. Their ultrastructure resembled that of the fibrocytes/fibroblasts and of the interstitial Cajal-like cells (ICLC). Double labelling and immunoelectro-microscopy demonstrated that they are unequivocally different to the ICC and, due to the similarities with the ICLC, we identified them as ICLC. The novelty of these results is that two types of interstitial cells are present in the GI muscle coat of humans: the ICC and the ICLC. We hypothesize a mechanical role for the septal ICLC, those at the myenteric plexus level and those bordering the muscle layers; a helping role in neurotransmission is proposed for the ICLC intercalated with the intramuscular ICC, possibly in spreading the slow waves generated by the ICC. Furthermore, the possibility that the ICLC represent the adult mesenchymal stromal cells able to guarantee the ICC renewal deserves to be considered.

Dynamic morphometric characterization of local connective tissue network structure in humans using ultrasound.

BACKGROUND: In humans, connective tissue forms a complex, interconnected network throughout the body that may have mechanosensory, regulatory and signaling functions. Understanding these potentially important phenomena requires non-invasive measurements of collagen network structure that can be performed in live animals or humans. The goal of this study was to show that ultrasound can be used to quantify dynamic changes in local connective tissue structure in vivo. We first performed combined ultrasound and histology examinations of the same tissue in two subjects undergoing surgery: in one subject, we examined the relationship of ultrasound to histological images in three dimensions; in the other, we examined the effect of a localized tissue perturbation using a previously developed robotic acupuncture needling technique. In ten additional non-surgical subjects, we quantified changes in tissue spatial organization over time during needle rotation vs. no rotation using ultrasound and semi-variogram analyses. RESULTS: 3-D renditions of ultrasound images showed longitudinal echogenic sheets that matched with collagenous sheets seen in histological preparations. Rank correlations between serial 2-D ultrasound and corresponding histology images resulted in high positive correlations for semi-variogram ranges computed parallel (r = 0.79, p < 0.001) and perpendicular (r = 0.63, p < 0.001) to the surface of the skin, indicating concordance in spatial structure between the two data sets. Needle rotation caused tissue displacement in the area surrounding the needle that was mapped spatially with ultrasound elastography and corresponded to collagen bundles winding around the needle on histological sections. In semi-variograms computed for each ultrasound frame, there was a greater change in the area under the semi-variogram curve across successive frames during needle rotation compared with no rotation. The direction of this change was heterogeneous across subjects. The frame-to-frame variability was 10-fold (p < 0.001) greater with rotation than with no rotation indicating changes in tissue structure during rotation. CONCLUSION: The combination of ultrasound and semi-variogram analyses allows quantitative assessment of dynamic changes in the structure of human connective tissue in vivo.

Synergistic effects of combined cytotoxic and apoptosis-inducing drugs on Tenon's capsule fibroblasts in vitro and in vivo.

BACKGROUND: Highly toxic antimetabolites have gained access to routine clinical use to modulate and reduce the amount of postoperative scarring following glaucomatous filtering procedures. It could be speculated that by combining two different antiproliferative substances with different mechanisms of action total amounts of the substances could be decreased and side effects reduced. METHODS: Twenty-two substances were tested that had antiproliferative effects by acting cytotoxically, inhibiting growth factors, or inducing apoptosis. With combinations of each two substances, cell culture experiments using 3T3 and human Tenon's capsule fibroblasts were performed evaluating cell toxicity, proliferation and migration, the extent of free radicals, and the amount of apoptosis (TUNEL, electron microscopy). The five most potent combinations were used in an animal experiment with rabbits performing filtering procedures. The extent of episcleral scarring was evaluated by histopathology. RESULTS: The results of the various assays revealed consistently strong effects in 5 of the 462 combinations. Of these five combinations, two were highly effective in the rabbit model. Substances with strong effects when applied in combination included staurosporine, mitomycin, and CD95L. CONCLUSIONS: We found synergistic effects in assays that evaluated different aspects of cell function. The amount of scarring in an animal experiment was inhibited to a level comparable with a high single dose of mitomycin. Combination therapy of two antiproliferative acting substances may be a promising concept.

Beyond the epithelium: cadherin function in fibrous connective tissues.

In fibrous connective tissues, fibroblasts are organized into syncytia, cellular networks that enable matrix remodeling and that are interconnected by intercellular adherens junctions (AJs). The AJs of fibroblasts are mediated by N-cadherin, a broadly expressed classical cadherin that is critically involved in developmental processes, wound healing and several diseases of mesenchymal tissues. In contrast to E-cadherin-dependent junctions of epithelia, the formation of AJs in fibrous connective tissues is relatively uncharacterized. Work over the last several years has documented an expanding list of molecules which function to regulate N-cadherin mediated junctions such as: Fer, PTP1B, cortactin, calcium, gelsolin, PIP5KIgamma, PIP2, and the Rho family of GTPases. We present an overview on the regulation of N-cadherin-mediated junction formation that highlights recent molecular advances in the field and rationalizes the roles of N-cadherin in connective tissue function.

Derivation of muscles of the Aristotle's lantern from coelomic epithelia.

Transmission electron microscopy was employed to study structural changes in the lantern muscles occurring during the transition from young to adult in the sea urchin Strongylocentrotus nudus. A comparative examination of four major lantern muscles (compass depressors, compass elevators, protractors and retractors) suggests that myogenesis involves four consecutive stages. At the initial stage, the muscles show the organization of a mesentery delimited by pseudostratified coelomic epithelia, which are composed of peritoneal cells spanning the whole height of each epithelium, and myoepithelial cells, which are clustered together to fill the interstices between the basal processes of the peritoneal cells. During the next stage, the clusters of myoepithelial cells partly "sink" into the underlying connective tissue. At the third stage of muscularization, the myoepithelial cells increase in size and further invade the underlying connective tissue so that the myoepithelium splits into an apical peritoneal layer and a deeper mass of myoepithelial cells immersed in the connective tissue. However, these two layers are connected by a continuous basal lamina. This is thus the first description of an intermediate developmental stage between pseudostratified myoepithelim and genuine echinoderm muscles. For such a myoepithelium, we propose the term "immersed myoepithelium". At the most advanced stage of myogenesis, the myocytes detach completely from the epithelium to form subepithelial muscle bundles. Myogenesis in the sea urchin takes a long time during which continuous myogenic differentiation occurs in the coelomic epithelium and the newly formed myocytes and associated neurons penetrate into the underlying connective tissue.