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Find video protocols related to scientific articles indexed in Pubmed.
Morphological and ultrastructural evaluation of the golden retriever muscular dystrophy trachea, lungs, and diaphragm muscle.
Microsc. Res. Tech.
PUBLISHED: 05-19-2014
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Duchenne muscular dystrophy (DMD) is a genetic disease, characterized by atrophy and muscle weakness. The respiratory failure is a common cause of early death in patients with DMD. Golden retriever muscular dystrophy (GRMD) is a canine model which has been extensively used for many advances in therapeutics applications. As the patients with DMD, the GRMD frequently died from cardiac and respiratory failure. Observing the respiratory failure in DMD is one of the major causes of mortality we aimed to describe the morphological and ultrastructural data of trachea, lungs (conductive and respiratory portion of the system), and diaphragm muscle using histological and ultrastructural analysis. The diaphragm muscle showed discontinuous fibers architecture, with different diameter; a robust perimysium inflammatory infiltrate and some muscle cells displayed central nuclei. GRMD trachea and lungs presented collagen fibers and in addition, the GRMD lungs showed higher of levels collagen fibers that could limit the alveolar ducts and alveoli distension. Therefore, the most features observed were the collagen areas and fibrosis. We suggested in this study that the collagen remodeling in the trachea, lungs, and diaphragm muscle may increase fibrosis and affect the trachea, lungs, and diaphragm muscle function that can be a major cause of respiratory failure that occur in patients with DMD. Microsc. Res. Tech. 77:857-861, 2014. © 2014 Wiley Periodicals, Inc.
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Seasonal variations cause morphological changes and altered spermatogenesis in the testes of viscacha (Lagostomus maximus).
Anim. Reprod. Sci.
PUBLISHED: 04-14-2014
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This study complements the previous investigations of the reproductive biology of male viscachas, a rodent of a seasonal Hystricognathi that exhibits photoperiod-induced morphological variations in the reproductive system. In the present study, a quantitative analysis of spermatogenesis was performed during the summer and the spring. Spermatogonial cells were analyzed to determine by immunolabelling for STRA8 and DAZL, which are essential for spermatogenesis. Six free-living male viscachas were captured, three animals in the summer during the period of reproductive activity and three animals in the spring during the period of testicular regression. The testes of the viscachas were collected and processed for light microscopy, macroscopic and immunochemical analyses. The germ and Sertoli cells present in the seminiferous tubules were quantitatively analyzed in each animal. The efficiency coefficient for spermatogonial mitosis, meiotic yield, overall spermatogenesis yield and Sertoli cell index, revealed that the Sertoli cells in male viscachas captured during the summer had a reduced capacity to structurally and nutritionally support the developing round spermatids compared with the male viscachas captured during the spring. The animals produced less sperm during the spring than the summer, suggesting a seasonal impact on spermatogenesis. Immunolabelling for STRA8 and DAZL was detected during summer and spring seasons. These results suggest that in seasonal rodents, such as the male viscachas, the photoperiod promotes significant changes in the testis and in the germ cell yield.
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Placentation and fetal membrane development in the South American coati, Nasua nasua (Mammalia, Carnivora, Procyonidae).
Reprod. Biol. Endocrinol.
PUBLISHED: 04-08-2014
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Placental research in carnivores has concentrated on domestic species, which have zonary, labyrinthine placentas with an endotheliochorial barrier. Although the coati, Nasua nasua, is a widely distributed species in South America, data on the development of the placenta and the fetal membranes in this species are very sparse.
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The effect of bone allografts combined with bone marrow stromal cells on the healing of segmental bone defects in a sheep model.
BMC Vet. Res.
PUBLISHED: 01-30-2014
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The repair of large bone defects is a major orthopedic challenge because autologous bone grafts are not available in large amounts and because harvesting is often associated with donor-site morbidity. Considering that bone marrow stromal cells (BMSC) are responsible for the maintenance of bone turnover throughout life, we investigated bone repair at a site of a critically sized segmental defect in sheep tibia treated with BMSCs loaded onto allografts. The defect was created in the mid-portion of the tibial diaphysis of eight adult sheep, and the sheep were treated with ex-vivo expanded autologous BMSCs isolated from marrow aspirates and loaded onto cortical allografts (n = 4). The treated sheep were compared with control sheep that had been treated with cell-free allografts (n = 4) obtained from donors of the same breed as the receptor sheep.
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Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation.
Stem Cells Cloning
PUBLISHED: 01-01-2014
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Amnion-derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases.
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Motor physical therapy affects muscle collagen type I and decreases gait speed in dystrophin-deficient dogs.
PLoS ONE
PUBLISHED: 01-01-2014
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Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.
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Development of bovine embryos derived from reproductive techniques.
Reprod. Fertil. Dev.
PUBLISHED: 12-29-2013
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Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.
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Reproductive stem cell differentiation: extracellular matrix, tissue microenvironment, and growth factors direct the mesenchymal stem cell lineage commitment.
Reprod Sci
PUBLISHED: 02-18-2013
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The mesenchymal stem cells (MSCs) have awakened interest in regenerative medicine due to its high capability to proliferate and differentiate in multiple specialized lineages under defined conditions. The reproductive system is considered a valuable source of MSCs, which needs further investigations. Many factors have been reported as critical for these cell lineage specification and determination. In this review, we discuss the main effects of extracellular matrix or tissue environment and growth factors in the cell lineage commitment, including the reproductive stem cells. The MSCs responses to culture medium stimuli or to soluble factors probably occur through several intracellular activation pathways. However, the molecular mechanisms in which the cells respond to these mechanical or chemical perturbations remain elusive. Recent findings suggest a synergic effect of microenvironment and soluble cell culture factors affecting cell differentiation. For future applications in cell therapy, protocols of reproductive MSCs differentiation must be established.
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A morphological analysis of the transition between the embryonic primitive intestine and yolk sac in bovine embryos and fetuses.
Microsc. Res. Tech.
PUBLISHED: 02-14-2013
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The yolk sac (YS) is the main source of embryonic nutrition during the period when the placenta has not yet formed. It is also responsible for hematopoiesis because the blood cells develop from it as part of the primitive embryonic circulation. The objective of this study was to characterize the transitional area between the YS and primitive gut using the techniques of light microscopy, transmission electron microscopy, and immunohistochemistry to detect populations of pluripotent cells by labeling with Oct4 antibody. In all investigated embryos, serial sections were made to permit the identification of this small, restricted area. We identified the YS connection with the primitive intestine and found that it is composed of many blood islands, which correspond to the vessels covered by vitelline and mesenchymal cells. We identified large numbers of hemangioblasts inside the vessels. The mesenchymal layer was thin and composed of elongated cells, and the vitelline endodermal membrane was composed of large, mono- or binucleated cells. The epithelium of the primitive intestine comprised stratified columnar cells and undifferentiated mesenchymal cells. The transitional area between the YS and the primitive intestine was very thin and composed of cells with irregular shapes, which formed a delicate lumen containing hemangioblasts. In the mesenchyme of the transitional area, there were a considerable number of small vessels containing hemangioblasts. Using Oct4 as a primary antibody, we identified positive cells in the metanephros, primordial gonad, and hepatic parenchyma as well as in YS cells, suggesting that these regions contain populations of pluripotent cells.
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Diaphragm morphology of guinea pig (Cavia porcellus).
Microsc. Res. Tech.
PUBLISHED: 01-19-2013
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The diaphragm is the main respiratory muscle. Along with other respiratory muscles, the diaphragm is responsible for the muscular contraction that generates the respiratory cycle and, as a consequence, the gaseous interchanges in the lungs. Guinea pigs (Cavia porcellus Linnaeus 1758) are largely used as experimental animals in many biology applications due to their easy management, low cost, and docile behavior. As the diaphragm exerts important effects on lung physiology and function, this study aimed at investigating the morphological characteristics of the muscle, through macroscopic, microscopic, and scanning electron microscopy to add reference data for future studies. We observed a "U"-shaped tendineous center and its morphology was similar to other mammals. These results cooperate with the descriptive and comparative anatomy of mammals, besides can be used as control data for areas of surgery and stem cells.
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Marsupial morphology of reproduction: South America opossum male model.
Microsc. Res. Tech.
PUBLISHED: 01-03-2013
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This study aims to describe the morphology of Didelphis sp. male genital organs (penis, testes, epididymis, ductus deferens, prostate, and bulbourethral gland). Ten male animals were used, eight for macroscopic and light microscopy analysis, and two for scanning electron microscopy. The testes and epididymis showed similarity to other eutherian mammals. The bifid penis showed the urethra ending in the medial region where the bifurcation begins, occurring in each segment extension of the urethral groove until the beginning of the glans. Histologically, the penis consists of a cavernous and spongy body, covered by stratified squamous epithelium with loose connective tissue. The urethra was lined by transitional stratified epithelium. In the prostate, prostatic segments were found consisting of tubular glands in a radial arrangement around the urethra, coated externally by a dense connective tissue associated with a relatively thick layer of smooth muscle arranged in two layers that surround the glandular tissue. The animals had three pairs of bulbourethral glands placed at the membranous and cavernous urethra junction with descending and parallel excretory ducts ending caudally in the urethral lumen.
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Bortezomib (PS-341) treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.
PLoS ONE
PUBLISHED: 01-01-2013
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Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NF?B and TGF-?1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of ?- and ?-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.
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Morphological characterization of the progenitor blood cells in canine and feline umbilical cord.
Microsc. Res. Tech.
PUBLISHED: 09-20-2011
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The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.
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Propolis and amnion reepithelialise second-degree burns in rats.
Burns
PUBLISHED: 05-13-2011
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Burns are serious consequences of trauma in terms of both imminent mortality and prolonged periods of morbidity. They are often accompanied by unsatisfactory cosmetic as well as functional and psychological outcomes. These complications emphasise the need for stronger efforts in achieving greater diversity and effectiveness in the treatment of skin burns. This study aimed to verify the effectiveness of gross and microscopic epidermal and dermal responses in the process of regenerative repair or healing of burns in rats that were treated either daily with 5% propolis ointment or by autologous amnion graft. Second-degree burns were inflicted in the neck region of female rats by contact with a hot metal (at 130 °C) for 5 s. Propolis treatment accelerated the process of tissue repair and led to decreased local inflammation, which indicates that treatment with propolis was successful in the initial period (7 days) and stimulated the production of collagen fibre (assessed by morphometry) in all the periods evaluated (14 and 21 days). Amnion treatment inhibited local inflammation (assessed macroscopically), stimulated local epithelial regeneration (assessed microscopically) and stimulated the production of collagen fibre (assessed by morphometry) in the days following burn. These treatments offer new therapeutic strategies for treating severe skin burns; these strategies may allow the minimisation of scar formation, a more rapid return of function and, ultimately, a better quality of life for burn patients.
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Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow.
Tissue Eng Part A
PUBLISHED: 05-03-2011
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During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM- and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.
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Characteristics of the equine embryo and fetus from days 15 to 107 of pregnancy.
Theriogenology
PUBLISHED: 04-13-2011
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In spite of numerous, substantial advances in equine reproduction, many stages of embryonic and fetal morphological development are poorly understood, with no apparent single source of comprehensive information. Hence, the objective of the present study was to provide a complete macroscopic and microscopic description of the equine embryo/fetus at various gestational ages. Thirty-four embryos/fetuses were aged based on their crown rump length (CRL), and submitted to macroscopic description, biometry, light and scanning microscopy, as well as the alizarin technique. All observed developmental changes were chronologically ordered and described. As examples of the main observed features, an accentuated cervical curvature was observed upon macroscopic examination in all specimens. In the nervous system, the encephalic fourth ventricle and the encephalic vesicles forebrain, midbrain, and hindbrain, were visualized from Day 19 (ovulation = Day 0). The thoracic and pelvic limbs were also visualized; their extremities gave rise to the hoof during development from Day 27. Development of other structures such as pigmented optical vesicle, liver, tail, cardiac area, lungs, and dermal vascularization started on Days 25, 25, 19, 19, 34, and 35, respectively. Light and scanning microscopy facilitated detailed examinations of several organs, e.g., heart, kidneys, lungs, and intestine, whereas the alizarin technique enabled visualization of ossification. Observations in this study contributed to the knowledge regarding equine embryogenesis, and included much detailed data from many specimens collected over a long developmental interval.
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The use of animal models for stroke research: a review.
Comp. Med.
PUBLISHED: 04-05-2011
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Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that improve knowledge of the pathology of stroke in both human and veterinary medicine.
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Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents.
J. Exp. Zool. B Mol. Dev. Evol.
PUBLISHED: 02-03-2011
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The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago.
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Placentation in Sigmodontinae: a rodent taxon native to South America.
Reprod. Biol. Endocrinol.
PUBLISHED: 02-03-2011
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Sigmodontinae, known as "New World rats and mice," is a large subfamily of Cricetidae for which we herein provide the first comprehensive investigation of the placenta.
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Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.
Cell Reprogram
PUBLISHED: 01-17-2011
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The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast?+?MII hemi-cytoplast?+ SC (G-I); IVF hemi-cytoplast?+?IVF hemi-cytoplast?+?SC (G-II); MII hemi-cytoplast?+?IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast?+?IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA?+?CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.
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Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results.
Acta Cir Bras
PUBLISHED: 02-09-2010
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Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH).
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Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.
Cloning Stem Cells
PUBLISHED: 09-16-2009
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Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.
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Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy.
Reprod. Biol. Endocrinol.
PUBLISHED: 03-30-2009
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Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities.
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Presumed normal ultrasonographic findings of the testis and epididymis of botos (Inia geoffrensis).
J. Zoo Wildl. Med.
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Fifteen live adult male botos, or Amazon river dolphins (Inia geoffrensis), were examined using ultrasonography during the yearly capture expedition, between October and November 2005, at the Mamirauá Sustainable Development Reserve, within the Brazilian Amazon (3 degrees S, 65 degrees W). All examinations were performed with a Sonosite 180 plus ultrasound unit in conjunction with a 2- to 5-MHz multifrequency transducer convex array 180 Plus/Elite-C60. Age and maturity estimates were determined considering the body length, weight, and external characteristics. In all examinations, the testes were discerned by the presence of a hyperechoic central line, called the mediastinum testis, a landmark for their identification during ultrasonography. No significant differences in echogenicity were detected on the ultrasonographic appearance of the testes among the studied animals. On adult male botos, apparent parenchymal nodulation of the testis was observed on scanning in most of the animals and probably constituted evidence of reproductive maturity. Using the color Doppler technique, blood flow was detected along the mediastinum testis that progressively decreased toward the periphery of this organ. Little blood flow could be identified by color Doppler. Power Doppler allowed better accuracy to identify testicular vessels, their topography, and their differentiation from adjacent structures. Ultrasonographic examination provides useful data for morphologic characterization of the botos testes. Examination using Doppler techniques was considered a valuable tool to evidence blood flow through the testicular parenchyma.
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Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice.
Acta Vet. Scand.
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The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.
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Placentation in the anteaters Myrmecophaga tridactyla and Tamandua tetradactyla (Eutheria, Xenarthra).
Reprod. Biol. Endocrinol.
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Since Xenarthra are serious candidates for being basal to Eutheria, their characteristics, e.g. the placental system, influence perceptions of evolution. However, in the subgroup containing the anteaters, data are very limited. The present study aims to elucidate the nature of the feto-maternal interface in the anteater placenta and to interpret these data within an evolutionary context.
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Kidney injury and cell therapy: Preclinical study.
Microsc. Res. Tech.
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The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst®. Nine agoutis males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 × 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agoutis as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG.
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Macroscopic and microscopic analysis of the tongue of the common opossum (Didelphis marsupialis).
Microsc. Res. Tech.
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We performed a macroscopic and microscopic study of the tongues of common opossums, Didelphis marsupialis, from South America. We studied two males and two females. We collected morphometric data on the tongue with precision calipers. For the light microscopy and scanning electron microscopy analyses, we fixed tissue fragments in 10% formaldehyde and 2.5% glutaraldehyde, respectively. The opossum tongues averaged 5.87 ± 0.20 cm in length, 3.27 ± 0.15 cm in width at the lingual body, and 3.82 ± 0.15 cm in width at the root. The mean thickness of the lingual body was 1.8 ± 0.1 cm, and the thickness of the root was 3.82 ± 0.15 cm. Sharp filiform papillae were scattered across the entire tongue; conical filiform papillae occurred on the lingual body and tongue tip; fungiform papillae were scattered among the filiform papillae on the lingual body and tongue tip; and there were three vallate papillae at the root of the tongue. We found two strands of papillary projections in the tongue root. Despite the low variability observed in the lingual papillae, the morphological data obtained in this study may be related to the opossums diverse food habits and the extensive geographic distribution of the species throughout America.
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Morphological observations of ampullae of lorenzini in Squatina guggenheim and S. occulta (Chondrichthyes, Elasmobranchii, Squatinidae).
Microsc. Res. Tech.
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We have conducted a morphological study of the ampullae of Lorenzini on two shark species from Squatina Genus. In both species, S. guggenheim and S. occulta, the ampullae were observed like small pores scattered in the head region similar to other species of the Chondrichthyes Class. However, differently of the other species a greatest density of ampullae of Lorenzini was observed along of the body surface. After fixation using 10% formaldehyde, the ampullae were removed and processed for light and scanning electron microscopy. Macroscopically, the two shark species differed by the presence of dorsal spines that appeared from the head to the first dorsal fin in S. guggenheim and were absent in S. occulta. Microscopically, there were no differences between the ampullae of Lorenzini channels in these two species. The wall of the ampulla was formed by a simple squamous epithelium. Bands of connective tissue, hyaline cartilage and collagen fibers were found between the ampulla and the skeletal striated muscle layer. Nerve branches responsible for conducting signal pulses to the central nervous system were visible between the muscle and connective tissue layers. Using scanning electron microscopy and histological analysis, we found that the channels were twisted and positioned parallel to the skin. The inside of the channels contained a large amount of a gelatinous secretion composed by polysaccharides. Therefore, we conclude that the morphological combination of extended distribution of the ampullae of Lorenzini and the body shape may represent an adaptation of these species to their way of life.
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Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells.
BMC Vet. Res.
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The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-?. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry.
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