RESUMO
Sex cord-stromal tumors (SCSTs), generally referred to as granulosa cell tumors (GCTs) or granulosa-theca cell tumors (GTCTs) in equids, show complex compositions and variable numbers of hormone-producing cells. These tumors can be difficult to diagnose, especially in early stages. Therefore, we tested a panel of antibodies for vimentin, smooth muscle actin, laminin, Ki-67, E-cadherin, calretinin, moesin, p-ezrin, AMH, and aromatase, markers used for tumor composition and classification, progression, and prognosis in human SCSTs, on an exemplary grapefruit-size equine GCT within the left ovary of a 13-year-old mare with stallion-like behavior and elevated testosterone levels in comparison with normal ovarian tissue. The tumor showed a low proliferation rate and prominent moesin and p-ezrin staining in granulosa cells. E-cadherin, calretinin, aromatase, and AMH are suggested to be potential markers for different cell components of equine SCSTs that can support tumor diagnosis and classification.
RESUMO
Purpose: The proper function of the tenocyte network depends on cell-matrix as well as intercellular communication that is mechanosensitive. Building on the concept that the etiopathogenic stimulus for tendon degeneration is the catabolic response of tendon cells to mechanobiologic under-stimulation, we studied the pericellular matrix rich in versican and its predominant proteolytic enzyme ADAMTS-1, as well as Connexin-43 (Cx43), a major gap junction forming protein in tendons, in stress-deprived rat tail tendon fascicles (RTTfs).Materials and Methods: RTTfs were stress-deprived for up to 7 days under tissue culture conditions. RT-qPCR was used to measure mRNA expression of versican, ADAMTS-1, and Cx43. Protein synthesis was determined using Western blotting and immunohistochemistry.Results: Stress-deprivation (SD) caused a statistically significant up-regulation of versican, ADAMTS-1, and Cx43 mRNA expression that was persistent over the 7-day test period. Western blot analysis and immunohistochemical assessment of protein synthesis revealed a marked increase of the respective proteins with SD. Inhibition of proteolytic enzyme activity with ilomastat prevented the increased versican degradation and Cx43 synthesis in 3 days stress-deprived tendons when compared with non-treated, stress-deprived tendons.Conclusion: In the absence of mechanobiological signaling the immediate pericellular matrix is modulated as tendon cells up-regulate their production of ADAMTS-1, and versican with subsequent proteoglycan degradation potentially leading to cell signaling cues increasing Cx43 gap junctional protein. The results also provide further support for the hypothesis that the cellular changes associated with tendinopathy are a result of decreased mechanobiological signaling and a loss of homeostatic cytoskeletal tension.
Assuntos
Conexina 43/metabolismo , Versicanas , Animais , Conexinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Tendões/metabolismo , Regulação para Cima , Versicanas/metabolismoRESUMO
OBJECTIVE: Transplantation of minor salivary glands (MSGs) to the conjunctiva is a treatment option for patients suffering from dry eye disease. As there is not enough information about labial and buccal MSGs in dogs, the aim of this study was to provide evidence of the presence of these glands and to investigate their spatial arrangement and excretory ducts. METHODS: The oral mucosa of the lower lip of 4 dogs and the whole lower jaw of 1 dog were used for histological and microCT analysis. Presence, number, volumes and the tissue depth of MSGs were assessed. RESULTS: Histological analysis showed that compact tubulo-acinar glands were located in the submucosal connective tissue. MicroCT images revealed that 9 to 21 MSGs were arranged in a single row at the level of the dental alveolae. The volume of the MSGs increased from rostral to caudal and the total volume of glandular tissue per animal ranged from 35.01 mm3 to 549.43 mm3 . The mean tissue depth of MSGs ranged from 0.57 mm to 1.37 mm (upper surface of glands) and between 1.43 mm and 3.09 mm (lower surface of the glands). Excretory ducts left the dorsal part of the glands and ran in dorso-rostral direction. CONCLUSIONS: The location, number and volume of the labial and buccal MSGs in the dog could be detected and described using microCT scans and histology. The present results can provide valuable information for future transplantation of labial MSGs as therapeutic measure against keratoconjunctivitis sicca.
Assuntos
Cães/anatomia & histologia , Glândulas Salivares/anatomia & histologia , Animais , Feminino , Processamento de Imagem Assistida por Computador , Masculino , Mucosa Bucal/anatomia & histologia , Glândulas Salivares Menores/anatomia & histologiaRESUMO
Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.
Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/lesões , Terapia Baseada em Transplante de Células e Tecidos/métodos , Osteoartrite/patologia , Regeneração/fisiologia , Proteínas de Fase Aguda/metabolismo , Animais , Células Cultivadas , Feto/fisiologia , Macrófagos/citologia , Células-Tronco Mesenquimais/metabolismo , Neutrófilos/citologia , Ovinos , Membrana Sinovial/citologia , Membrana Sinovial/lesões , Membrana Sinovial/metabolismoRESUMO
Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.
Assuntos
Matriz Extracelular/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Regeneração , Tendinopatia/metabolismo , Tendões/fisiologia , Animais , Matriz Extracelular/patologia , Feminino , Feto , Humanos , Ovinos , Tendinopatia/patologiaRESUMO
Hyperphosphatemic conditions such as chronic kidney disease are associated with severe muscle wasting and impaired life quality. While regeneration of muscle tissue is known to be reliant on recruitment of myogenic progenitor cells, the effects of elevated phosphate loads on this process have not been investigated in detail so far. This study aims to clarify the direct effects of hyperphosphatemic conditions on skeletal myoblast differentiation in a murine in vitro model. C2C12 murine muscle progenitor cells were supplemented with phosphate concentrations resembling moderate to severe hyperphosphatemia (1.4-2.9 mmol/l). Phosphate-induced effects were quantified by RT-PCR and immunoblotting. Immunohistochemistry was performed to count nuclear positive cells under treatment. Cell viability and metabolic activity were assessed by XTT and BrdU incorporation assays. Inorganic phosphate directly induced ERK-phosphorylation in pre-differentiated C2C12 myoblast cells. While phosphate concentrations resembling the upper normal range significantly reduced Myogenin expression (- 22.5%, p = 0.015), severe hyperphosphatemic conditions further impaired differentiation (Myogenin - 61.0%, p < 0.0001; MyoD - 51.0%; p < 0.0001). Analogue effects were found on the protein level (Myogenin - 42.0%, p = 0.004; MyoD - 25.7%, p = 0.002). ERK inhibition strongly attenuated phosphate-induced effects on Myogenin expression (p = 0.002). Metabolic activity was unaffected by the treatments. Our data point to a phosphate-induced inhibition of myoblast differentiation without effects on cell viability. Serum phosphate levels as low as the upper normal serum range significantly impaired marker gene expression in vitro. Investigation of cellular effects of hyperphosphatemia may help to better define serum cutoffs and modify existing treatment approaches of phosphate binders, especially in patients at risk of sarcopenia.
Assuntos
Expressão Gênica/genética , Mioblastos Esqueléticos/metabolismo , Fosfatos/metabolismo , Animais , Diferenciação Celular , CamundongosRESUMO
Cells use different cell adhesion and communication structures to promote tissue development, maintenance of tissue integrity as well as repair and regenerative processes. Another recently discovered way of information exchange is long-distance thin cellular processes called nanotubes (NTs), mainly studied in vitro. Information on the existence and relevance of NTs in vivo is sparse. Building on two references which hint at the potential existence of longitudinally directed cell processes resembling NTs, we investigated tendons from young (3 weeks) and adult (9 weeks, 4 and 8 months) Fisher rats. Whole mounts of rat tail tendon fascicles (RTTfs) and sections of Achilles, flexor, extensor and patellar tendons were stained with Deep Red plasma membrane and DAPI nuclear stain and immunolabelled with Connexin43 (Cx43). In addition, 3-D reconstruction of serial semithin sections and TEM was used to verify the presence of NTs. We were able to demonstrate NTs as straight thin longitudinal processes (Ø 100-500 nm) reaching up to several 100 µm in length, mainly originating from lateral sheet-like cell processes or cell bodies in all tendon types investigated. NTs were observed to distend between tenocyte rows at the same level but also connect cells of different rows, thus leading to a complex 3-D cellular scaffold. Shorter NTs connected lateral cell sheets of tenocytes in the same row, omitting one or two cells. In addition, we detected links or potential branching of NTs. Cx43 immunostaining for the detection of gap junctions revealed Cx43-positive foci at the end-to-end contacts of tenocyte cell bodies as well as along their contacting sheet-like processes. Only rarely, we found clear Cx43 signals at their potential contact points between NTs and tendon cells as well as along the course of NTs, and most NTs appeared completely devoid of Cx43 signals. Therefore, we conclude that NTs in tendons could have a twofold function: long-distance communication as well as stabilization of a mechanically challenged tissue. From in vitro studies it is known that NTs allow intercellular transmission of various cell components, offering potential protective effects for the respective tissue. Further studies on functional properties of NTs in tendons under changing mechanical loading regimens are required in the future. The fact that NTs are present in tendons may necessitate the reconsideration of our traditional understanding of cell-to-cell communication.
Assuntos
Comunicação Celular/fisiologia , Tendões/citologia , Tenócitos/citologia , Animais , Adesão Celular/fisiologia , Células Cultivadas , Conexina 43/metabolismo , Ratos , Ratos Endogâmicos F344 , Tendões/metabolismo , Tenócitos/metabolismoRESUMO
Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology-driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro-morphology and gene expression of newborn, young and old horses. Age-related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genes collagen type 1 (Col1), Col3, Col5, tenascin-C, decorin, tenomodulin, versican, scleraxis and cartilage oligomeric matrix protein were investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin-C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older, decorin expression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age-related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age.
Assuntos
Envelhecimento/fisiologia , Colágeno/genética , Decorina/genética , Expressão Gênica , Proteínas de Membrana/genética , Tendões/metabolismo , Fatores Etários , Animais , Colágeno/metabolismo , Decorina/metabolismo , Cavalos , Proteínas de Membrana/metabolismoRESUMO
OBJECTIVE: The morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation-, differentiation-, stem cell-associated markers and cell junction proteins were assessed. METHODS: Corneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E-cadherin, ß-catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium. RESULTS: The height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea-specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E-cadherin, ß-catenin, and Cx43 revealed a similar apico-lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface. CONCLUSIONS: This study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.
Assuntos
Células Epiteliais/fisiologia , Epitélio Corneano/anatomia & histologia , Epitélio Corneano/química , Cavalos/anatomia & histologia , Imuno-Histoquímica/veterinária , Envelhecimento , Animais , Anticorpos/imunologia , Epitélio Corneano/citologia , Regulação da Expressão Gênica/fisiologia , Células-Tronco/fisiologiaRESUMO
αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid-5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co-localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor-αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium-conserving hormone in the kidney.
Assuntos
Cálcio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Rim/metabolismo , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Membrana Celular/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase , Proteínas Imediatamente Precoces/metabolismo , Proteínas Klotho , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease-induced substances is highly desirable. METHODS: Bone marrow-derived equine MSCs were transduced with a lentiviral vector expressing interleukin-1 receptor antagonist (IL-1Ra) gene under the control of an inducible nuclear factor-kappa B-responsive promoter and IL-1Ra production upon pro-inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)-1ß] was analysed. To assess the biological activity of the IL-1Ra protein that was produced and the therapeutic effect of IL-1Ra-expressing MSCs (MSC/IL-1Ra), cytokine-based two- and three-dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real-time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin-1ß, interleukin-6, interleukin-8, matrix metalloproteinase-1 and matrix metalloproteinase-13. RESULTS: A dose-dependent increase in IL-1Ra expression was found in MSC/IL-1Ra cells upon TNFα administration, whereas stimulation using IL-1ß did not lead to IL-1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL-1Ra protein present in the conditioned medium from MSC/IL-1Ra cells blocks OA onset in cytokine-treated equine chondrocytes and co-cultivation of MSC/IL-1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes. CONCLUSIONS: Thus, pro-inflammatory cytokine induced IL-1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.
Assuntos
Citocinas/farmacologia , Expressão Gênica/efeitos dos fármacos , Doenças dos Cavalos/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/genética , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Engenharia Genética/métodos , Terapia Genética/métodos , Doenças dos Cavalos/patologia , Doenças dos Cavalos/terapia , Cavalos , Humanos , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Lentivirus/genética , Masculino , Células-Tronco Mesenquimais/citologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Osteoartrite/patologia , Osteoartrite/terapia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Recent advances in human fascia research have shed new light on the role of the fascial network in movement perception and coordination, transmission of muscle force, and integrative function in body biomechanics. Evolutionary adaptations of equine musculoskeletal apparatus that assure effective terrestrial locomotion are employed in equestrianism, resulting in the wide variety of movements in performing horses, from sophisticated dressage to jumping and high-speed racing. The high importance of horse motion efficiency in the present-day equine industry indicates the significance of scientific knowledge of the structure and physiology of equine fasciae. In this study, we investigated the structure and innervation of the deep fascia of the equine forelimb by means of anatomical dissection, histology and immunohistochemistry. Macroscopically, the deep fascia appears as a dense, glossy and whitish lamina of connective tissue continuous with its fibrous reinforcements represented by extensor and flexor retinacula. According to the results of our histological examination, the general structure of the equine forelimb fascia corresponds to the characteristics of the human deep fasciae of the limbs. Although we did find specific features in all sample types, the general composition of all examined fascial tissues follows roughly the same scheme. It is composed of dense, closely packed collagen fibers organized in layers of thick fibrous bundles with sparse elastic fibers. This compact tissue is covered from both internal and external sides by loosely woven laminae of areolar connective tissue where elastic fibers are mixed with collagen. Numerous blood vessels running within the loose connective tissue contribute to the formation of regular vascular network throughout the compact layer of the deep fascia and retinacula. We found nerve fibers of different calibers in all samples analyzed. The fibers are numerous in the areolar connective tissue and near the blood vessels but scarce in the compact layers of collagen. We did not observe any Ruffini, Pacini or Golgi-Mazzoni corpuscles. In conclusion, the multilayered composition of compact bundles of collagen, sparse elastic fibers in the deep fascia and continuous transition into retinacula probably facilitate resistance to gravitational forces and volume changes during muscle contraction as well as transmission of muscle force during movement. However, further research focused on innervation is needed to clarify whether the deep fascia of the equine forelimb plays a role in proprioception and movement coordination.
Assuntos
Fáscia/inervação , Membro Anterior/anatomia & histologia , Cavalos/anatomia & histologia , Animais , Feminino , MasculinoRESUMO
It is crucial but challenging to keep physiologic conditions during the cultivation of 3D cell scaffold constructs for the optimization of 3D cell culture processes. Therefore, we demonstrate the benefits of a recently developed miniaturized perfusion bioreactor together with a specialized incubator system that allows for the cultivation of multiple samples while screening different conditions. Hence, a decellularized bone matrix was tested towards its suitability for 3D osteogenic differentiation under flow perfusion conditions. Subsequently, physiologic shear stress and hydrostatic pressure (HP) conditions were optimized for osteogenic differentiation of human mesenchymal stem cells (MSCs). X-ray computed microtomography and scanning electron microscopy (SEM) revealed a closed cell layer covering the entire matrix. Osteogenic differentiation assessed by alkaline phosphatase activity and SEM was found to be increased in all dynamic conditions. Furthermore, screening of different fluid shear stress (FSS) conditions revealed 1.5 mL/min (equivalent to â¼10 mPa shear stress) to be optimal. However, no distinct effect of HP compared to flow perfusion without HP on osteogenic differentiation was observed. Notably, throughout all experiments, cells cultivated under FSS or HP conditions displayed increased osteogenic differentiation, which underlines the importance of physiologic conditions. In conclusion, the bioreactor system was used for biomaterial testing and to develop and optimize a 3D cell culture process for the osteogenic differentiation of MSCs. Due to its versatility and higher throughput efficiency, we hypothesize that this bioreactor/incubator system will advance the development and optimization of a variety of 3D cell culture processes.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Osteogênese , Perfusão/instrumentação , Materiais Biocompatíveis/química , Diferenciação Celular , Células Cultivadas , Desenho de Equipamento , Feminino , Humanos , Pressão Hidrostática , Pessoa de Meia-Idade , Porosidade , Engenharia Tecidual/instrumentação , Alicerces Teciduais/químicaRESUMO
BACKGROUND: Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease-modifying therapies of osteoarthritis, cell-based treatments offer a promising new approach. Owing to their paracrine, differentiation and self-renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regulated by natural disease-dependent occuring substances, is of high interest. METHODS: Bone marrow-isolated equine MSCs were stably transduced with an HIV-1 based lentiviral vector expressing the luciferase gene under control of an inducible nuclear factor κB (NFκB)-responsive promoter. Marker gene expression was analysed by determining luciferase activity in transduced cells stimulated with different concentrations of interleukin (IL)-1ß or tumour necrosis factor (TNF)α. RESULTS: A dose-dependent increase in luciferase expression was observed in transduced MSCs upon cytokine stimulation. The induction effect was more potent in cells treated with TNFα compared to those treated with IL-1ß. Maximum transgene expression was obtained after 48 h of stimulation and the same time was necessary to return to baseline luciferase expression levels after withdrawal of the stimulus. Repeated cycles of induction allowed on-off modulation of transgene expression without becoming refractory to induction. The NFκB-responsive promoter retained its inducibility also in chondrogenically differentiated MSC/Luc cells. CONCLUSIONS: The results of the present study demonstrate that on demand transgene expression from the NFκB-responsive promoter using naturally occurring inflammatory cytokines can be induced in undifferentiated and chondrogenically differentiated equine MSCs. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Expressão Gênica , Engenharia Genética/métodos , Inflamação/genética , Células-Tronco Mesenquimais/metabolismo , Transgenes/genética , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Citocinas/farmacologia , Cavalos , Humanos , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The vascularization pattern of the equine stifle joint is insufficiently described in the literature, even though there is a growing need for knowledge of the exact blood supply, as (i) arthroscopy and endoscopic surgery techniques are increasingly performed in horses and (ii) ex vivo models of menisci need nutrient supply that mimic the in vivo situation. The aim of this study was to describe the vessels involved in the stifle joint supply and the exact branching pattern of the popliteal artery. Colored latex was injected into the arteries of nine pelvic limbs of equine cadavers (n = 6) to evaluate the occurrences, variations and approximate diameters of vessels that supplied the stifle joints. Next to a branch of the saphenous and descending genicular arteries, eleven branches of the popliteal artery could be described in horses that feed the vascular network of the stifle joint. With a focus on the blood supply of the menisci, a vascularization map was created to show the main influx to these intra-articular structures in detail. These findings are potentially of great importance to both clinicians in preparation of best-suited incisions for arthroscopy and researchers designing new approaches for meniscal studies and choosing suitable animal models.
RESUMO
INTRODUCTION: Regulation of phosphate homeostasis is essential for mineralization and enchondral ossification. Fibroblast growth factor 23 (FGF23) and its obligatory co-receptor Klotho (KL) play a key role in this process by influencing both renal phosphate reabsorption and vitamin D metabolism. In disease, excessive action of FGF23 leads to hypophosphatemic rickets, while its deficiency causes tumoral calcinosis. Although osteocytes and osteoblasts are widely seen as the primary source of FGF23 under physiological conditions, the origin of systemic FGF23 remains controversial. In this study, we investigated the expression of FGF23 and KL in porcine growth plate cartilage, adjacent tissues, and parenchymal tissues. MATERIALS AND METHODS: Tissue samples were obtained from 4- to 6-week-old piglets. mRNA expression was quantified by real-time PCR and normalized to 18S rRNA. Immunohistochemical staining was performed for FGF23, KL, collagen type X, and FGF receptor 1. Growth plate chondrocyte subpopulations were acquired by collagenase digestion of growth plate explants and subsequent density gradient centrifugation. RESULTS: We could detect both FGF23 and KL mRNA and protein in growth plate chondrocytes. FGF23 expression was mainly found in hypertrophic and resting chondrocytes. Furthermore, significant expression of both genes was observed in bone, liver, and spleen. CONCLUSION: These data challenge previous expression analyses, in particular theories of bone as the exclusive source of FGF23. Moreover, significant expression of FGF23 and KL within the growth plate and adjacent tissues imply a potential local role of FGF23 in chondrocyte differentiation and tissue mineralization.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Lâmina de Crescimento/metabolismo , Animais , Osso e Ossos/metabolismo , Cartilagem Articular/metabolismo , Centrifugação com Gradiente de Concentração , Colágeno Tipo X/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucuronidase/genética , Lâmina de Crescimento/citologia , Imuno-Histoquímica , Proteínas Klotho , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem , Sus scrofaRESUMO
OBJECTIVE: To investigate the density and distribution of conjunctival goblet cells (GC) and study the anatomy and microscopic characteristics of glands associated with the eye in chinchillas (Chinchilla Laniger). PROCEDURE: 12 chinchillas were included in the study. Conjunctiva (divided into four regions), eyelids, and glands were embedded in paraffin wax, sectioned, stained, and analyzed. RESULTS: Highest GC densities were found in the palpebral region of the nasal and temporal conjunctiva of both eyelids (GC index: 25.1-18.2%), and lowest densities, in the bulbar and marginal region of the nasal and temporal conjunctiva of both eyelids (GC index: 1.5-0.0%). Meibomian glands extend along the entire length of both eyelids, and the whole glandular complex broadens toward the temporal canthus. This is macroscopically visible through the conjunctiva. The openings of the Meibomian glands are macroscopically not discernible. The light pink, smooth, and crescent-shaped lacrimal gland lies next to the aforementioned broadened part of the Meibomian glands in the temporal canthus. The whitish, 0.9-cm-long, smooth Harderian gland is firmly attached to the posterior part of the globe and extends nasally from the optic nerve to the equator. Furthermore, chinchillas possess two lacrimal puncta, situated on the inner conjunctival surface of both eyelids near the medial canthus. A pigmented lacrimal canaliculus originates from each punctum. The vestigial nictitating membrane is supported by a hyaline cartilage and is pigmented at its free margin. CONCLUSIONS: Chinchillas possess a Harderian gland, a lacrimal gland, and Meibomian glands. The GC density in the nasal and temporal palpebral conjunctiva is higher than in guinea pigs.
Assuntos
Chinchila/anatomia & histologia , Túnica Conjuntiva/citologia , Células Caliciformes/citologia , Glândula de Harder/anatomia & histologia , Aparelho Lacrimal/anatomia & histologia , Glândulas Tarsais/anatomia & histologia , Animais , Olho/anatomia & histologia , Olho/citologia , Células Caliciformes/fisiologia , Glândula de Harder/fisiologia , Aparelho Lacrimal/fisiologia , Glândulas Tarsais/fisiologiaRESUMO
BACKGROUND/OBJECTIVE: The purpose of this study was to establish a reliable and reproducible method to isolate and cultivate canine corneal epithelial and stromal cells (cCECs and cCSCs). The cells were subsequently used for in vitro testing of topically applied diluted povidone iodine (PVI). METHODS: Corneas of dogs, euthanized for reasons unrelated to this study, were used to collect primary cCECs and cCSCs. Corneas were enzymatically digested and explants obtained using biopsy punches. Epithelial and stromal explants were separately taken into culture. Cell proliferation and migration was evaluated after incubation of cCECs and cCSCs with PVI in different concentrations (1, 2, or 5%) and with different exposure times (1, 3, or 10 min). RESULTS: Solely incubation of 4 mm diameter corneal epithelial explants in a 48-well culture plate in full medium led to sufficient growth of cCECs. Up to four passages were achieved with a cell density of 10,000-20,000 cells/cm2 after dissociation of cells in trypsin for 8 min. Cell detachment and passaging for cCSCs were possible with almost every cornea and explant. Canine CSCs were less sensitive to PVI in all concentrations and over time than cCECs. Epithelial and stromal cell proliferation and migration decreased with increasing exposure times and PVI concentrations across all groups. CONCLUSIONS: The described method is a straightforward and sound way to isolate and cultivate cCECs and cCSCs in vitro. Basic information on cCEC and cCSC migration and proliferation after incubation with PVI, was gathered. The results may provide a step towards an optimal preoperative antisepsis protocol for ophthalmic surgery in future.
Assuntos
Córnea , Povidona-Iodo , Animais , Proliferação de Células , Cães , Células Epiteliais , Povidona-Iodo/farmacologia , Células EstromaisRESUMO
This systematic review highlights the similarities and variations in Ossa cordis prevalence, histology and anatomical location between differing veterinary species and in humans. In addition, it also identifies associated factors such as aging and cardiovascular disease for each species in relation to functional roles and developmental mechanisms that these bone structures may play. The potential functions of Ossa cordis are presented, ranging from aiding cardiac contraction and conduction, providing cardiac structure, and protecting components of the heart, through to counteracting high mechanical stress. Furthermore, this review discusses the evidence and rationale behind the theories regarding the formation and development of Ossa cordis in different veterinary species and in people.
Assuntos
Doenças Cardiovasculares , Coração , Humanos , Animais , Osso e Ossos , Doenças Cardiovasculares/veterináriaRESUMO
The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues.