RESUMEN
Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.
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Displasia Ventricular Derecha Arritmogénica , Adipogénesis/fisiología , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología , Muerte Súbita Cardíaca/patología , Humanos , Lípidos , Células del Estroma/metabolismoRESUMEN
During long bone development the original cartilaginous model in mammals is replaced by bone, but at the long bone endings the avascular articular cartilage remains. Before the articular cartilage attains structural maturity it undergoes reorganization, and molecules such as vascular endothelial growth factor (VEGF) and endostatin could be involved in this process. VEGF attracts blood vessels, whereas endostatin blocks their formation. The present study therefore focused on the spatio-temporal localization of these two molecules during the development of the articular cartilage. Furthermore, we investigated the distribution of the chondro/osteoclasts at the chondro-osseous junction of the articular cartilage with the subchondral bone. Mice served as our animal model, and we examined several postnatal stages of the femur starting with week (W) 4. Our results indicated that during the formation of the articular cartilage, VEGF and endostatin had an overlapping localization. The former molecule was, however, down-regulated, whereas the latter was uniformly intensely localized until W12. At the chondro-osseous junction, the number of tartrate-resistant acid phosphatase (TRAP)-positive chondro/osteoclasts declined with increasing age. Until W3 the articular cartilage was not well organized but at W8 it appeared structurally mature. At that time only a few TRAP cells were present, indicative of a low resorptive activity at the chondro-osseous junction. Subsequently, we examined the metaphyseal growth plate that is closed when skeletal maturity is attained. Within its hypertrophic zone, localization of endostatin and VEGF was observed until W6 and W8, respectively. At the chondro-osseous junction of the growth plate, chondro/osteoclasts remained numerous until W12 to allow for its complete resorption. According to former findings, VEGF is critical for a normal skeleton development, whereas endostatin has almost no effect on this process. In conclusion, our findings suggest that both VEGF and endostatin play a role in the structural reorganization of the articular cartilage and endostatin may be involved in the maintenance of its avascularity. In the growth plate, however, endostatin does not appear to counteract VEGF, allowing vascular invasion of hypertrophic cartilage and bone growth.
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Cartílago Articular/citología , Cartílago Articular/metabolismo , Endostatinas/metabolismo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Fosfatasa Ácida , Animales , Condrocitos/citología , Fémur/citología , Fémur/crecimiento & desarrollo , Fémur/metabolismo , Placa de Crecimiento/crecimiento & desarrollo , Inmunohistoquímica , Isoenzimas , Ratones , Modelos Animales , Osteoclastos/citología , Fosfatasa Ácida TartratorresistenteRESUMEN
The bones are of mesenchymal or ectomesenchymal origin, form the skeleton of most vertebrates, and are essential for locomotion and organ protection. As a living tissue they are highly vascularized and remodelled throughout life to maintain intact. Bones consist of osteocytes entrapped in a mineralized extracellular matrix, and via their elaborated network of cytoplasmic processes they do not only communicate with each other but also with the cells on the bone surface (bone lining cells). Bone tissue develops through a series of fine-tuned processes, and there are two modes of bone formation, referred to either as intramembranous or endochondral ossification. In intramembranous ossification, bones develop directly from condensations of mesenchymal cells, and the flat bones of the skull, the clavicles and the perichondral bone cuff develop via this process. The bones of the axial (ribs and vertebrae) and the appendicular skeleton (e.g. upper and lower limbs) form through endochondral ossification where mesenchyme turns into a cartilaginous intermediate with the shape of the future skeletal element that is gradually replaced by bone. Endochondral ossification occurs in all vertebrate taxa and its onset involves differentiation of the chondrocytes, mineralization of the extracellular cartilage matrix and vascularization of the intermediate, followed by disintegration and resorption of the cartilage, bone formation, and finally - after complete ossification of the cartilage model - the establishment of an avascular articular cartilage. The epiphyseal growth plate regulates the longitudinal growth of the bones, achieved by a balanced proliferation and elimination of chondrocytes, and the question whether the late hypertrophic chondrocytes die or transform into osteogenic cells is still being hotly debated. The complex processes leading to endochondral ossification have been studied for over a century, and this review aims to give an overview of the histological and molecular events, arising from the long bones' (e.g. femur, tibia) development. The fate of the hypertrophic chondrocytes will be discussed in the light of new findings obtained from cell tracking studies.
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Cartílago , Placa de Crecimiento , Animales , Desarrollo Óseo , Condrocitos , Osteogénesis , CráneoRESUMEN
In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio-temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.
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Desarrollo Óseo/fisiología , Huesos/metabolismo , Cartílago/metabolismo , Epífisis/crecimiento & desarrollo , Epífisis/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Huesos/citología , Cartílago/citología , Ratones , Factores de TiempoRESUMEN
To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumor-induced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor alpha (TNFalpha) antagonist etanercept. In naive mice, exogenous TNFalpha evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNFalpha enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen-activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNFalpha as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.
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Carcinoma/complicaciones , Carcinoma/metabolismo , Hiperalgesia/etiología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Capsaicina/farmacología , Células Cultivadas , Etanercept , Eliminación de Gen , Miembro Posterior , Calor , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Hiperalgesia/prevención & control , Inmunoglobulina G/farmacología , Ratones , Trasplante de Neoplasias , Neuronas Aferentes/efectos de los fármacos , Nociceptores/efectos de los fármacos , Nociceptores/fisiopatología , Técnicas de Placa-Clamp , Receptores del Factor de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Canales Catiónicos TRPV/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells. Considering the well-known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1-MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.
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Fosfatasa Ácida/análisis , Fémur/crecimiento & desarrollo , Isoenzimas/análisis , Macrófagos/fisiología , Metaloproteinasa 14 de la Matriz/análisis , Osteogénesis/fisiología , Fosfatasa Ácida/metabolismo , Animales , Biomarcadores/análisis , Desarrollo Óseo/fisiología , Cartílago/enzimología , Cartílago/crecimiento & desarrollo , Condrocitos/enzimología , Histocitoquímica , Inmunohistoquímica , Isoenzimas/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Osteoblastos/enzimología , Fosfatasa Ácida TartratorresistenteRESUMEN
In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.
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Desarrollo Óseo , Cartílago/crecimiento & desarrollo , Cartílago/fisiología , Fosfatasa Ácida/genética , Animales , Cartílago/irrigación sanguínea , Endopeptidasas/genética , Placa de Crecimiento/fisiología , Humanos , Mamíferos , Metaloproteasas/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: We herein investigate critical ischemia times and the effect of novel preservation solutions such as new histidine-tryptophan-ketoglutarate (HTK-N) and TiProtec on the individual tissues of a rat limb isograft. METHODS: Orthotopic hind-limb transplantations were performed in male Lewis rats after 2 hours, 6 hours, or 10 hours of cold ischemia (CI). Limbs were flushed and stored in HTK-N, TiProtec, HTK, or saline solution. Muscle, nerve, vessel, skin, and bone samples were procured on day 10 for histology, immunohistochemistry, confocal and electron microscopy, and quantitative real-time polymerase chain reaction analysis. RESULTS: Histomorphology of the muscle showed a mainly perivascular inflammatory infiltrate, fibrotic degeneration, and neovascularization after 6 hours and 10 hours of CI. However, centrally aligned nuclei observed in muscle fibers suggest for muscle regeneration in these samples. In addition to Wallerian degeneration, nerve injury was significantly aggravated (P = 0.032) after prolonged CI. Proinflammatory and regulatory cytokines were most significantly upregulated after 2-hour CI. Our data suggest no superiority of novel perfusates HTK-N and TiProtec in terms of tissue preservation, compared with HTK and saline. CONCLUSIONS: Limiting CI time for less than 6 hours is the most significant factor to reduce tissue damage in vascularized tissue transplantation. Signs of muscle regeneration give rise that ischemic muscle damage in limb transplantation might be reversible to a certain extent.
Asunto(s)
Trasplante Óseo/efectos adversos , Isquemia Fría/efectos adversos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/trasplante , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Trasplante de Piel/efectos adversos , Animales , Trasplante Óseo/métodos , Citoprotección , Regulación de la Expresión Génica , Glucosa/farmacología , Miembro Posterior/metabolismo , Miembro Posterior/ultraestructura , Isoinjertos , Masculino , Manitol/farmacología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Animales , Desarrollo de Músculos/efectos de los fármacos , Cloruro de Potasio/farmacología , Procaína/farmacología , Ratas Endogámicas Lew , Reacción en Cadena en Tiempo Real de la Polimerasa , Regeneración/efectos de los fármacos , Trasplante de Piel/métodos , Factores de Tiempo , Supervivencia Tisular/efectos de los fármacos , Degeneración WallerianaRESUMEN
Balance orientation depends on the precise operation of the vestibular end organs and the vestibular ganglion neurons. Previous research on the assemblage of the neuronal network in the developing fetal vestibular organ has been limited to data from animal models. Insights into the molecular expression profiles and signaling moieties involved in embryological development of the human fetal inner ear have been limited. We present an investigation of the cells of the vestibular end organs with specific focus on the hair cell differentiation and innervation pattern using an uninterrupted series of unique specimens from gestational weeks 8-12. Nerve fibers positive for peripherin innervate the entire fetal crista and utricle. While in rodents only the peripheral regions of the cristae and the extra-striolar region of the statolithic organs are stained. At week 9, transcription factors PAX2 and PAX8 were observed in the hair cells whereas PAX6 was observed for the first time among the supporting cells of the cristae and the satellite glial cells of the vestibular ganglia. Glutamine synthetase, a regulator of the neurotransmitter glutamate, is strongly expressed among satellite glia cells, transitional zones of the utricle and supporting cells in the sensory epithelium. At gestational week 11, electron microscopic examination reveals bouton contacts at hair cells and first signs of the formation of a protocalyx at type I hair cells. Our study provides first-hand insight into the fetal development of the vestibular end organs as well as their pattern of innervation by means of immunohistochemical and EM techniques, with the aim of contributing toward our understanding of balance development.
RESUMEN
Studies on the formation of neuronal structures of the human cochlea are rare, presumptively, due to the difficult accessibility of specimens, so that most investigations are performed on mouse models. By means of immunohistochemical and transmission electron microscopic techniques, we investigated an uninterrupted series of unique specimens from gestational week 8 to week 12. We were able to demonstrate the presence of nerve fibers in the prosensory domain at gestational week 8, followed by afferent synaptogenesis at week 11. We identified PAX2 as an early marker for hair cell differentiation. Glutamine synthetase-positive peripheral glial cells occurred at the beginning of week 8. Transcription factor MAF B was used to demonstrate maturation of the spiral ganglion neurons. The early expression of tyrosine hydroxylase could be assessed. This study provides insights in the early assembly of the neural circuit and organization in humans.
Asunto(s)
Oído Interno/crecimiento & desarrollo , Oído Interno/inervación , Adulto , Oído Interno/metabolismo , Feto , Humanos , Inmunohistoquímica , Factor de Transcripción MafB/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción PAX2/metabolismo , Periferinas/metabolismo , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Sinapsis/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECTIVES: Tissue-engineered xenografts represent a promising treatment option in heart valve disease. However, inflammatory response leading to graft failure and incomplete in vitro repopulation with recipient cells remain challenging. Shock waves (SWs) were shown to modulate inflammation and to enhance re-epithelialization. We therefore aimed to investigate whether SWs could serve as a feasible adjunct to tissue engineering. METHODS: Porcine aortic pieces were decellularized using sodium deoxycholate and sodium dodecylsulphate and implanted subcutaneously into C57BL/6 mice (n = 6 per group). The treatment (shock wave therapy, SWT) group received SWs (0.1 mJ/mm(2), 500 impulses, 5 Hz) for modulation of inflammatory response directly after implantation; control animals remained untreated (CTR). Grafts were harvested 72 h and 3 weeks after implantation and analysed for inflammatory cytokines, macrophage infiltration and polarization, osteoclastic activity and calcification. Transmission electron microscopy (TEM) was performed. Endothelial cells (ECs) were treated with SWs and analysed for macrophage regulatory cytokines. In an ex vivo experimental set-up, decellularized porcine aortic valve conduits were reseeded with ECs with and without SWT (0.1 mJ/mm(2), 300 impulses, 3 Hz), fibroblasts as well as peripheral blood mononuclear cells (all human) and tested in a pulsatile flow perfusion system for cell coverage. RESULTS: Treated ECs showed an increase of macrophage migration inhibitory factor and macrophage inflammatory protein 1ß, whereas CD40 ligand and complement component C5/C5a were decreased. Subcutaneously implanted grafts showed increased mRNA levels of tumour necrosis factor α and interleukin 6 in the treatment group. Enhanced repopulation with recipient cells could be observed after SWT. Augmented macrophage infiltration and increased polarization towards M2 macrophages was observed in treated animals. Enhanced recruitment of osteoclastic cells in proximity to calcified tissue was found after SWT. Consequently, SWT resulted in decreased areas of calcification in treated animals. The reseeding experiment revealed that fibroblasts showed the best coverage compared with other cell types. Moreover, SW-treated ECs exhibited enhanced repopulation compared with untreated controls. CONCLUSIONS: SWs reduce the calcification of subcutaneously implanted decellularized xenografts via the modulation of the acute macrophage-mediated inflammatory response and improves the in vitro repopulation of decellularized grafts. It may therefore serve as a feasible adjunct to heart valve tissue engineering.
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Aorta/metabolismo , Válvula Aórtica/metabolismo , Bioprótesis , Calcinosis/patología , Prótesis Valvulares Cardíacas , Ondas de Choque de Alta Energía/uso terapéutico , Animales , Aorta/citología , Aorta/patología , Aorta/efectos de la radiación , Válvula Aórtica/citología , Válvula Aórtica/patología , Válvula Aórtica/efectos de la radiación , Citocinas/análisis , Enfermedades de las Válvulas Cardíacas , Masculino , Ratones , Ratones Endogámicos C57BL , PorcinosRESUMEN
Sorting of native (unpermeabilized) SVF-cells from human subcutaneous (s)WAT for cell surface staining (cs) of DLK1 and CD34 identified three main populations: ~10% stained cs-DLK1+/cs-CD34-, ~20% cs-DLK1+/cs-CD34+dim and ~45% cs-DLK1-/cs-CD34+. FACS analysis after permeabilization showed that all these cells stained positive for intracellular DLK1, while CD34 was undetectable in cs-DLK1+/cs-CD34- cells. Permeabilized cs-DLK1-/cs-CD34+ cells were positive for the pericyte marker α-SMA and the mesenchymal markers CD90 and CD105, albeit CD105 staining was dim (cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31-). Only these cells showed proliferative and adipogenic capacity. Cs-DLK1+/cs-CD34- and cs-DLK1+/cs-CD34+dim cells were also α-SMA+ but expressed CD31, had a mixed hematopoietic and mesenchymal phenotype, and could neither proliferate nor differentiate into adipocytes. Histological analysis of sWAT detected DLK1+/CD34+ and DLK1+/CD90+ cells mainly in the outer ring of vessel-associated stroma and at capillaries. DLK1+/α-SMA+ cells were localized in the CD34- perivascular ring and in adventitial vascular stroma. All these DLK1+ cells possess a spindle-shaped morphology with extremely long processes. DLK1+/CD34+ cells were also detected in vessel endothelium. Additionally, we show that sWAT contains significantly more DLK1+ cells than visceral (v)WAT. We conclude that sWAT has more DKL1+ cells than vWAT and contains different DLK1/CD34 populations, and only cs-DLK1-/cs-CD34+/CD90+/CD105+dim/α-SMA+/CD45-/CD31- cells in the adventitial vascular stroma exhibit proliferative and adipogenic capacity.
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Tejido Adiposo Blanco/citología , Antígenos CD34/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Células del Estroma/metabolismo , Actinas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Proteínas de Unión al Calcio , Diferenciación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Microscopía Fluorescente , Persona de Mediana Edad , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Células del Estroma/citologíaRESUMEN
Complete series of semithin sections are imperative for 3-D reconstruction, but with traditional microtomy techniques it is difficult and time-consuming to trace stained and labeled structures. In the present study we introduce a method for making and collecting ribbons of semithin sections with a new, commercial available diamond knife (histo-jumbo-diamond knife, Diatome AG, Biel, Switzerland). The special feature of the diamond knife is the large water bath (boat) into which a glass slide can be dipped. The method has distinct advantages and the handling is simple. The resin block is trimmed into a truncated pyramid. Contact glue is applied to the leading face of the pyramid, which makes sections stick together to form a ribbon. Following sectioning, the ribbons are mounted onto glass slides and aligned in parallel. Stretching out and drying the ribbons on a hot plate is the final step of the method. Major advantages of this method are the perfect alignment of sections with identical orientation of structures, the completeness of series, and the significant saving of time. This facilitates tracing of stained and labeled structures, yielding quick 3-D reconstruction. Semithin sections can be cut from 0.5 to 2 micro m and several ribbons can be mounted side by side onto the slide. Two examples are presented to illustrate the advantages of the method.
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Diamante , Microtomía/instrumentación , Animales , Diamante/química , Microtomía/métodos , Retina/citología , Caracoles , PorcinosRESUMEN
A detailed study of so-called communicating cartilage canals, which penetrate deeply up into the lower hypertrophic zone of the epiphyseal growth plate in the embryonic chicken femur (E20), was carried out with the aim to clarify whether or not these canals are involved in the bone-forming process. In addition, we examined the manner in which cartilage canals are formed and compare the present data with our previous data. The canals were investigated by means of light microscopy, electron microscopy, immunohistochemistry (VEGF, VEGFR2/Flk1, type I collagen), and 3D reconstruction. Some communicating canals deeply penetrate into the upper hypertrophic zone where they terminate, showing electron-dense cells at their end. Subcellular characteristics of these cells are hardly detectable and we suppose that they undergo cell death. Other canals pass down deeper into the lower hypertrophic zone. The upper segment of these canals is composed of capillaries, mesenchymal cells, and macrophage-like cells. Precursors of osteoblasts are adjacent to the canals. The lower segment of communicating canals is composed of bone matrix or osteoid, which contains type I collagen fibrils and cells having the typical subcellular features of osteoblasts. No vessels are found in these segments. Immunohistochemistry shows that the matrix of the canals labels positively for type I collagen. In addition, staining with sirius red demonstrates that bone matrix is formed in these parts. We assume that the osteoblast-like cells of the lower segments of communicating canals originate either from mesenchymal cells or even from hypertrophic chondrocytes. Our immunohistochemical data also reveal that vascular endothelial growth factor (VEGF) and the corresponding receptor VEGFR2/Flk1 (VEGF receptor 2/Flk1) are localized in cartilage canals of the reserve zone, the proliferative zone, and the hypertrophic zone. The receptor is found in the endothelial cells of the vessels. Furthermore, VEGF is present in hypertrophic chondrocytes. The results of our study suggest that cartilage canals penetrate actively into the cartilage anlage and that bone is formed in the lower segments of the communicating canals where no vessels are detectable.
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Desarrollo Óseo/fisiología , Cartílago/embriología , Embrión de Pollo/crecimiento & desarrollo , Fémur/embriología , Placa de Crecimiento/embriología , Animales , Embrión de Pollo/metabolismo , Embrión de Pollo/ultraestructura , Colágeno Tipo I/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía Electrónica , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model. METHODS: Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy. RESULTS: Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr. CONCLUSION: Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.
Asunto(s)
Extremidades/trasplante , Soluciones Preservantes de Órganos/química , Preservación de Órganos/instrumentación , Daño por Reperfusión/diagnóstico , Adenosina/química , Alopurinol/química , Animales , Isquemia Fría , Relación Dosis-Respuesta a Droga , Extremidades/irrigación sanguínea , Glucosa/química , Glutatión/química , Inflamación , Insulina/química , Masculino , Manitol/química , Microscopía Confocal , Microscopía Electrónica de Transmisión , Músculo Esquelético/patología , Preservación de Órganos/métodos , Cloruro de Potasio/química , Procaína/química , Rafinosa/química , Ratas , Ratas Endogámicas Lew , Nervio Ciático/patología , Factores de TiempoRESUMEN
The organogenesis of the male human urethra is still a subject of controversy. Although many studies have been conducted, the mechanisms of urethral development still need further investigation to clarify questions concerning the sequences in its development. Our aim has been to elucidate the spatiotemporal distribution of relevant immunohistochemical indicators during the development of the male urethral epithelium and its adjacent mesenchyme. Therefore, we analyzed male human embryos and foetuses between the 6th and 15th week after fertilization using histological and immunohistochemical methods. Monoclonal antibodies raised against cytokeratins (CKs) 7, 8, 13 and 17 as well as Ki67, E-Cadherin, p63, uroplakin III, smooth muscle actin (SMA) and vimentin were applied. Our results revealed that epithelial differentiation starts prior to the rupture of the cloacal membrane. At weeks (W) 8-9 the epithelium became transitional over the whole length of the elongating urethra. The urothelial staining pattern of uroplakin III receded continually, and, at the end of W 11, it had receded in proximal direction to the bladder neck comparable to the epithelial appearance in adults. The urogenital sinus epithelium provided the Wolffian duct with p63-positive cells, leading to the suggestion that the development of the male inner genitals requires a cellular stimulus by this very epithelium. CK 17-positive cells, which were described as epithelial stem cells, could be found in the extending urethral plate. This facilitates new insight into the pathogenesis and treatment of hypospadias, which is one of the most common malformations in newborn males.
Asunto(s)
Queratinas/metabolismo , Proteínas de la Membrana/metabolismo , Uretra/embriología , Uretra/fisiología , Vimentina/metabolismo , Biomarcadores/metabolismo , Humanos , Recién Nacido , Masculino , Análisis Espacio-Temporal , Distribución TisularRESUMEN
The female urethra has often been neglected in previous studies on the development of the human urogenital system. Our aim has been to reach a consensus on the organogenesis of the female urethra and the vagina with respect to interactions between the epithelia with different evolutionary origins. Therefore we tried to clarify open questions on the spatiotemporal distribution of molecular markers raised against mesenchymal and epithelial structures within the developing human female urethra. Furthermore, we draw comparisons regarding gender-specific aspects in urethral development. To this effect, we used molecular markers such as different cytokeratins (CKs), p63, Ki67, uroplakin III, E-cadherin, vimentin, smooth muscle actin (SMA), cleaved caspase 3 and paired box gene 2 (PAX 2) to phenotype developmental changes. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay was additionally performed to reveal apoptosis. We examined different gestational stages starting from week (W) 8 until W 15. Immunohistochemistry showed a distinct staining pattern for p63 and CK17, both markers for stem cells, ensuing from the urogenital sinus (UGS) proceeding into the Muellerian duct (MD). This was observed throughout development and might be a stimulus for the formation of the vaginal anlagen that derive from the MD. In the attachment area of the MD we detected a conglomeration of cells with different embryonic origins. The epithelium of the UGS became transitional at W 9 after fertilization, and the differentiation advanced in a cranial to caudal direction. The paraurethral glands showed a slightly different staining profile than the urethral epithelium, which may be able to explain why carcinomas of these structures display various histological appearances. In addition, we could show that during the development of the female urogenital system the primary incidence is the formation of the urethra. This is followed by the establishment of the vagina, which clearly depends on the proper differentiation of the UGS/urethra.
Asunto(s)
Epitelio/embriología , Uretra/embriología , Biomarcadores/análisis , Muerte Celular , Diferenciación Celular , Epitelio/fisiología , Femenino , Feto/citología , Edad Gestacional , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Recién Nacido , Mesodermo/citología , Embarazo , Uretra/fisiología , Sistema Urogenital/embriologíaRESUMEN
Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácido Glutámico/toxicidad , Pérdida Auditiva/complicaciones , Pérdida Auditiva/patología , Neurotoxinas/toxicidad , Sepsis/complicaciones , Sepsis/patología , Animales , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Caspasa 3/metabolismo , Cóclea/enzimología , Cóclea/patología , Cóclea/fisiopatología , Cóclea/ultraestructura , Potenciales Evocados Auditivos del Tronco Encefálico , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/patología , Células Ciliadas Auditivas Internas/ultraestructura , Pérdida Auditiva/fisiopatología , Inmunohistoquímica , Ligadura , Ratones , Ratones Endogámicos C57BL , Punciones , Sepsis/fisiopatología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.
Asunto(s)
Fosfatasa Ácida/metabolismo , Desarrollo Óseo , Epífisis/enzimología , Epífisis/crecimiento & desarrollo , Isoenzimas/metabolismo , Animales , Biomarcadores/metabolismo , Médula Ósea/crecimiento & desarrollo , Matriz Ósea/citología , Matriz Ósea/enzimología , Calcificación Fisiológica , Cartílago Articular/citología , Cartílago Articular/crecimiento & desarrollo , Epífisis/citología , Fémur/citología , Fémur/enzimología , Fémur/crecimiento & desarrollo , Placa de Crecimiento/citología , Inmunohistoquímica , Ratones , Tamaño de los Órganos , Osteoblastos/citología , Osteoblastos/enzimología , Osteogénesis , Fosfatasa Ácida TartratorresistenteRESUMEN
The mouse mutant wavy tail Tg(Col1a1-lacZ)304ng was created through transgene insertion and exhibits defects of the vertebral column. Homozygous mutant animals have compressed tail vertebrae and wedge-shaped intervertebral discs, resulting in a meandering tail. Delayed closure of lumbar neural arches and lack of processus spinosi have been observed; these defects become most prominent during the transition from cartilage to bone. The spina bifida was resistant to folic acid treatment, while retinoic acid administration caused severe skeletal defects in the mutant, but none in wild type control animals. The transgene integrated at chromosome 11 band D, in an area of high gene density. The insertion site was located between the transcription start sites of the Rpl23 and Lasp1 genes. LASP1 (an actin binding protein involved in cell migration and survival) was found to be produced in resting and hypertrophic chondrocytes in the vertebrae. In mutant vertebrae, temporal and spatial misexpression of Lasp1 was observed, indicating that alterations in Lasp1 transcription are most likely responsible for the observed phenotype. These data reveal a yet unappreciated role of Lasp1 in chondrocyte differentiation during cartilage to bone transition.