Isolation and characterization of neural stem cells from fetal canine spinal cord.

Neurogenesis in adult mammals occurs mainly in the subventricular and subgranular areas of the brain, but there are also reports of its occurrence in the spinal cord. In a study on rats, neural stem cells and neuroprogenitor cells could be obtained through primary spinal cord culture, but there are no studies on these cells in canine species, to date. Dogs represent an appropriate animal model for studies on neurogenesis and neurological disorders. In addition, they are animals of great affective value, and the therapeutic use of neural stem cells can represent a breakthrough in regenerative veterinary medicine. Therefore, this study aimed to determine a protocol for the isolation, culture, and characterization of neural and neuroprogenitor stem cells derived from the spinal cord of canine fetuses. The cells were isolated from spinal cord fragments and cultured in serum-free culture medium supplemented with EGF and FGF-2 growth factors. These cells were observed daily by optical microscopy to analyze their morphological characteristics. From the third day in vitro, it was possible to observe translucent cell groupings, similar to the neurospheres, which approximately ranged from 50 µm to 200 µm at seven days in vitro. Throughout the culture period, the neurospheres developed ribbons in their periphery that migrated and communicated with other neurospheres. RT-PCR revealed that the cells expressed the characteristic genes SOX2, NESTIN, and GFAP. In addition to gene expression, the cells were phenotypically marked in the immunofluorescence assay for the proteins Nestin, GFAP, and ß-tubulin III, characterizing them as neurospheres. Our results suggest that the spinal cord may be a source of neural stem cells and neural progenitor cells in canine fetuses. These cells may be an interesting option for neurogenesis and neuroregenerative therapy studies.

Neurochemical properties of neurospheres infusion in experimental-induced seizures.

Cell replacement through neural stem cells has been a promising alternative therapy for neurodegenerative diseases. It was evaluated the possible protect and/or prevent role of neurospheres in experimental models of epilepsy by the use of biomarkers of oxidative stress and histopathological analysis. After 1 h of the epileptic inductions by pilocarpine, pentylenotetrazole and picrotoxin, rats were infused with a suspension of 2 × 106 cells/0.25 mL, marked with Qtracker® 655, via caudal vein. In the control group epilepsy was not induced, but received the cell infusion under the same conditions of other groups. After 30 days, the rats were euthanized, and the removal of the brain was proceeded to later perform the assays oxidative stress and histopathology analysis. Thiobarbituric acid and nitrite levels were elevated in epileptic groups treated with neurospheres, and the levels of reduced glutathione, superoxide dismutase and catalase were reduced when compared to non-treated groups. The performance of oxidative enzymes from pilocarpine group treated with neurospheres showed slight increase. Histopathological evaluation observed distribution of neurospheres throughout the brain tissue, with viable cells and in process of differentiation in the pilocarpine group, but with differentiation and regeneration compromised in epilepsy by picrotoxin and pentylenetetrazole due to a microenvironment of oxidative stress. Neural stem cell therapy has a promising potential for protection in the pilocarpine epilepsy model, suggesting that the antioxidant system of neurospheres could reduce oxidative damage generated by seizure.

Behavior and biocompatibility of rabbit bone marrow mesenchymal stem cells with bacterial cellulose membrane.

BACKGROUND: Tissue engineering has been shown to exhibit great potential for the creation of biomaterials capable of developing into functional tissues. Cellular expansion and integration depends on the quality and surface-determinant factors of the scaffold, which are required for successful biological implants. The objective of this research was to characterize and evaluate the in vitro characteristics of rabbit bone marrow mesenchymal stem cells (BM-MSCs) associated with a bacterial cellulose membrane (BCM). We assessed the adhesion, expansion, and integration of the biomaterial as well as its ability to induce macrophage activation. Finally, we evaluated the cytotoxicity and toxicity of the BCM. METHODS: Samples of rabbit bone marrow were collected. Mesenchymal stem cells were isolated from medullary aspirates to establish fibroblast colony-forming unit assay. Osteogenic, chondrogenic, and adipogenic differentiation was performed. Integration with the BCM was assessed by scanning electron microscopy at 1, 7, and 14 days. Cytotoxicity was assessed via the production of nitric oxide, and BCM toxicity was assessed with the MTT assay; phagocytic activity was also determined. RESULTS: The fibroblastoid colony-forming unit (CFU-F) assay showed cells with a fibroblastoid morphology organized into colonies, and distributed across the culture area surface. In the growth curve, two distinct phases, lag and log phase, were observed at 15 days. Multipotentiality of the cells was evident after induction of osteogenic, chondrogenic, and adipogenic lineages. Regarding the BM-MSCs' bioelectrical integration with the BCM, BM-MSCs were anchored in the BCM in the first 24 h. On day 7 of culture, the cytoplasm was scattered, and on day 14, the cells were fully integrated with the biomaterial. We also observed significant macrophage activation; analysis of the MTT assay and the concentration of nitric oxide revealed no cytotoxicity of the biomaterial. CONCLUSION: The BCM allowed the expansion and biointegration of bone marrow progenitor cells with a stable cytotoxic profile, thus presenting itself as a biomaterial with potential for tissue engineering.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury.

PURPOSE:: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. METHODS:: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. RESULTS:: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. CONCLUSION:: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Rabbit olfactory stem cells. Isolation protocol and characterization.

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Greater rhea (Rhea americana) external morphology at different stages of embryonic and fetal development.

Knowledge of wild species embryonic development is important for their maintenance in captivity or the wild. The objective of the present study was to characterize the external morphology and define the biometry of greater rhea embryos and fetuses at different stages of development. A total of 41 embryos and fetuses were analyzed to describe their external morphology using a stereoscopic microscope. The crown-rump (CR), total length (TL), cephalocaudal length (CCL), biparietal diameter (BPD), beak, humerus and tibio-tarsal lengths were measured by digital pachymeter, millimetric scale ruler and cotton thread. The weight of the embryos and fetuses was measured on digital scales. The greater rhea embryos at 5, 6 and 7 days incubation presented a "C" shape. At 9, 10 and 11 days the eyes were big and pigmented. At 11, 12 and 13 days the eyelid covered more than half the eye, resulting in an oval slit. In 14 and 15 day-old embryos, the skin was still thin and the ribs evident, but at 18 days this structure was thicker. In embryos at 21 and 27 days of development closed eyelids were observed forming an eyelid slit, and the eye ball was less pronounced at 27 days. Weight gain presented an exponential growth curve, while measurements such as TL, DBP, beak, humerus and tibio-tarsal length had linear growth over time. Thus it was possible to characterize the greater rhea embryos and fetuses at several incubation ages using their external morphology and morphometric analyses.

Collared pecary (Tayassu tajacu) as a new model of renal ischemic injury induced by clamping the renal artery.

PURPOSE: The use of the collared peccary as an experimental model for ischemic nephropathy. METHODS: A total of 12 collared peccary (Tayassu tajacu) was used and ischemic nephropathy was induced in six of these animals that constituted the experimental group (G1) while the other six formed the control group (G2). Ischemic nephropathy was induced surgically by partial occlusion of the left renal artery. The disease course was assessed by hematological tests, serum chemistry, urinalysis, ultrasound (US) and doppler ultrasound function of the renal artery before induction, and at five, 10, 15 and 20 days after surgery. Twenty days after the occlusion, unilateral nephrectomy and histopathological examination were performed to assess renal morphology. RESULTS: Statistical analysis by Fischer's test showed a significant difference (p<0.05) between the control group and the experimental group. The histopathological examination showed glomerular, tubular and interstitial lesions. In the experimental group, 83.3% (5 /6) showed moderate renal lesions and only 16.7% (1/6) were classified with no lesions. The ultrasound examination of the right kidney presented statistical difference between day 5 and day 10 post occlusion. CONCLUSION: The collared peccary as a good experimental model for ischemic renal disease, because it could be manipulated during the research time without death, with health conditions that permit any subsequent procedure for disease therapy.

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil.

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury

Abstract Purpose: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. Methods: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. Results: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. Conclusion: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Serum biochemistry in hystricomorpha: agouti (Dasyprocta prymnolopha) during pregnancy / Bioquímica sérica em hystricomorpha: cutia (Dasyprocta prymnolopha, WAGLER, 1831) durante o período gestacional

The aim of this estudy was to establish the levels of serum total protein, albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium, phosphorus, urea, creatinine, bilirubin and glucose during pregnancy in agoutis. Animals: Twelve pregnant agouti from the Center for the Study and Preservation of Wild Animals (CSPWA) of the Federal University of Piauí (UFPI) were used in this research. After identification of the estrus, the day of the coverage was confirmed by means of vaginal cytology with the visualization of spermatozoa (day zero) and confirmation of pregnancy by ultrasonographic examination after 15 days. Blood samples were collected by lateral saphenous vein puncture after physical restraint, every 10 days until the end of pregnancy, for biochemical analyzes. A completely randomized experimental design was used and the means compared by the Duncan test at 5% probability using the SAS (Statistical Analysis System). The results of the biochemical analysis of total protein, albumin, globulin, urea, creatinine, calcium, phosphorus, serum ALT, glucose, AST, total bilirubin, direct bilirubin and indirect bilirubin in pregnant agouti (Dasyprocta prymnolopha) did not differ when compared to nonpregnant females. The serum biochemical levels during pregnancy in agoutis, except for calcium and phosphorus, were unchanged compared to those found in the non-pregnant adult animal, as occurs in other species. The changes during pregnancy reflect the physiology and biology of wild species, elucidating information about the biochemical parameters during pregnancy, thus characterizing the animal as a benchmark for comparisons with other species, extolling its importance both for nature conservation and production in capivity.

O estudo objetivou estabelecer os níveis séricos de proteínas totais, albumina, globulina, Alanina Aminotransferase (ALT), Aspartato Aminotransferase (AST), cálcio, fósforo, ureia, creatinina, bilirrubina e glicose durante a gestação em cutias. A pesquisa foi desenvolvida utilizando-se 12 cutias fêmeas criadas no Núcleo de Estudos, Produção e Preservação de Animais Silvestres da Universidade Federal do Piauí. Após a identificação do estro, o dia da cobertura foi confirmado por meio de citologia vaginal com a visualização de espermatozoides (dia zero) e confirmação da gestação por exame ultrassonográfico após 15 dias. Confirmada a gestação, foram coletados 03 mL de sangue mediante punção da veia pudenda interna, após contenção física, a cada 10 dias, até o final da gestação. Foi feita a análise de variância para um delineamento inteiramente casualizado com teste de Duncan para comparação das médias a 5% de probabilidade utilizando-se do programa estatístico SAS (Statistical Analysis System). Os resultados obtidos por meio da análise bioquímica de proteína total, albumina, globulina, ureia, creatinina, cálcio, fósforo, ALT séricas, glicose, aspartato aminotransferase (AST), bilirrubina total, bilirrubina direta e bilirrubina indireta de cutias gestantes (Dasyprocta prymnolopha) diferem de forma absoluta quando comparados a fêmeas não gestantes. Os níveis bioquímicos séricos durante a gestação em cutias, com exceção do cálcio, fósforo, sofrem alterações comparadas ao animal adulto não prenhe, como ocorre em outras espécies. Os níveis nas cutias gestantes sofrem alterações de acordo com o tempo de gestação, com maiores mudanças no período inicial e final da prenhez. As mudanças durante a gravidez refletem a fisiologia e a biologia da espécie silvestre, elucidando informações sobre os parâmetros bioquímicos durante a gestação, caracterizando o animal como referência para comparações com outras espécies, exaltando a importância tanto para sua conservação quanto para a sua produção em cativeiro.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep.

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Morphological analysis of peripheral blood cells of Chelonoidis carbonaria (Spix, 1824) / Análise morfológica das células do sangue periférico de Chelonoidis carbonaria (Spix, 1824)

This present work describe the peripheral blood cell morphology from Chelonoids carbonaria. To do this were used ten animal specimens clinically healthy, six female and four male, submitted to peripheral blood collect by jugular vein. Blood was collected to prepare blood smears, without the use of anticoagulants. The slides were stained and analyzed microscopically to describe the cell morphology. The mature erythrocytes had an ellipsoid shape and a grain-free nucleus; immature ones were circular. The leukocytes, granulocytes and agranulocytes were also circular. The heterophils had cytoplasmic granules with various elongated shapes, and the eosinophils had a uniform round shape. The basophils had highly dense basophilic granules, stained in blue; the granules were irregularly arranged and also inside the nucleus. The lymphocytes were circular with a large circular nucleus. The thrombocytes were small, with basophilic staining and a small cytoplasm (the nucleus occupied almost the entire cell). The morphological results found in this study are consistent with cell types of other chelonians.

O presente estudo descreve a morfologia dos constituintes celulares do sangue periférico de Chelonoides carbonaria. Para tanto, 10 espécimes adultos, sendo seis fêmeas e quatro machos, clinicamente saudáveis foram submetidos à coleta de sangue periférico através da veia jugular. O sangue foi recolhido para preparar esfregaços sanguíneos, sem a utilização de anticoagulantes. As lâminas foram coradas e analisadas microscopicamente para descrever a morfologia da célula. Os eritrócitos maduros têm a forma elipsóide e apresentam núcleo central sem granulações; nas fases imaturas mostram-se arredondados. Os leucócitos, granulócitos e agranulócitos, também são circulares. Os heterófilos possuem grânulos citoplasmáticos com várias formas alongadas e nos eosinófilos são uniformes e arredondados. Os basófilos possuem grânulos altamente densos e basofílicos, corados em azul; os grânulos são dispostos de forma irregular e também no interior do núcleo. Os linfócitos são circulares com um grande núcleo circular. Os trombócitos são pequenos, com coloração basofílica e citoplasma escasso (o núcleo ocupa quase toda a célula). Os resultados encontrados nesta pesquisa são compatíveis com a morfologia encontrada nesses tipos celulares em outros quelônios.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results.

PURPOSE: 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). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Collared Pecary (tayassu tajacu) as a new model of renal ischemic injury induced by clamping the renal artery

PURPOSE: The use of the collared peccary as an experimental model for ischemic nephropathy. METHODS: A total of 12 collared peccary (Tayassu tajacu) was used and ischemic nephropathy was induced in six of these animals that constituted the experimental group (G1) while the other six formed the control group (G2). Ischemic nephropathy was induced surgically by partial occlusion of the left renal artery. The disease course was assessed by hematological tests, serum chemistry, urinalysis, ultrasound (US) and doppler ultrasound function of the renal artery before induction, and at five, 10, 15 and 20 days after surgery. Twenty days after the occlusion, unilateral nephrectomy and histopathological examination were performed to assess renal morphology. RESULTS: Statistical analysis by Fischer's test showed a significant difference (p<0.05) between the control group and the experimental group. The histopathological examination showed glomerular, tubular and interstitial lesions. In the experimental group, 83.3% (5 /6) showed moderate renal lesions and only 16.7% (1/6) were classified with no lesions. The ultrasound examination of the right kidney presented statistical difference between day 5 and day 10 post occlusion. CONCLUSION: The collared peccary as a good experimental model for ischemic renal disease, because it could be manipulated during the research time without death, with health conditions that permit any subsequent procedure for disease therapy. .

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied. .

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep / Protocolos para obtenção e isolamento de células tronco mesenquimais de duas fontes em ovinos

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

OBJETIVO: Testar diferentes protocolos para o isolamento de células tronco a partir de sangue de cordão umbilical e tecido adiposo de ovinos. MÉTODOS: Foram utilizadas cinco amostras de sangue de cordão umbilical e cinco amostras de tecido adiposo perirrenal de 10 fêmeas de ovelha. A coleta das amostras foi realizada através de procedimento cirúrgico para coleta do material de forma mais asséptica possível. Foram realizados três protocolos de isolamento e cultivo das células-tronco do cordão umbilical e quatro protocolos para o isolamento e cultivo das células-tronco de gordura de ovinos RESULTADOS: Somente um dos protocolos utilizados para o isolamento das células-tronco de cordão umbilical foi efetivo. Dos quatro protocolos utilizados para isolamento das células-tronco de gordura, da mesma forma, apenas um obteve sucesso. Foi realizado o ensaio de unidades formadoras de colônias destas células, sendo contadas 58 colônias ao final de sete dias. Na citometria de fluxo essas células mostraram-se positivas para CD44 e negativas para CD38, CD45, CD41/61. Estas células apresentaram curva de crescimento com fases de LOG, LAG e PLATEAU bem definidas, características das curvas de crescimento das células-tronco de origem mesenquimal. CONCLUSÕES: O isolamento e cultivo das células-tronco mesenquimais do cordão umbilical de ovinos é de difícil realização, exigindo maiores ensaios e estudos profundos. Células tronco do tecido adiposo de ovelhas demonstraram características mesenquimais, de acordo com a curva de crescimento, habilidade de formação de colônias, células com morfologia fibroblastóide e reação positiva ao anticorpo CD44.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results / Sucesso no transplante de células tronco mesenquimais em ovinos com osteonecrose induzida da cabeça do fêmur: resultados preliminares

PURPOSE: 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). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

OBJETIVO: Verificar os efeitos das células-tronco mesenquimais da medula óssea de ovinos e da polpa dentária imatura humana em ovinos com osteonecrose induzida, da cabeça do fêmur. MÉTODOS: Oito ovelhas foram distribuídas em três grupos experimentais. O primeiro grupo foi composto por quatro animais com osteonecrose da cabeça do fêmur induzida por etanol através da descompressão central, que não receberam nenhum tratamento. O segundo e o terceiro grupo, cada um composto por dois animais, receberam transplante heterólogo de células tronco mesenquimais de ovinos e polpa dentária imatura humana seis semanas após a indução da osteonecrose da cabeça do fêmur, respectivamente. Em ambos os grupos experimentais as células foram transplantadas sem o uso de drogas imunossupressoras. RESULTADOS: Os achados demonstram que as células-tronco mesenquimais injetadas na cabeça do fêmur se encontravam viáveis após o transplante no novo sítio e proliferaram em pouco tempo. Os dados histológicos sugerem que a regeneração óssea nos animais transplantados com polpa dentária imatura humana foi mais rápida do que nos animais submetidos somente a descompressão central. CONCLUSÃO: O transplante de células tronco mesenquimais na osteonecrose da cabeça do fêmur induzida em ovinos através da técnica de descompressão central é um procedimento seguro, e aparentemente favorece a regeneração óssea de tecidos lesados.

Contaminação por enteroparasitas em amostras de alfaces (Lactuca sativa) de hortas produtoras de verduras da ilha de São Luís, MA / Contamination for parasites in samples of lettuces (Lactuca sativa) of producing of vegetables of the island of São Luís, MA

Um grande número de enfermidades entéricas são veiculadas através de verduras contaminadas. Este estudo teve como objetivo a verificação da contaminação por enteroparasitas em amostras de alfaces (Lactuca sativa) provenientes de hortas da ilha de São Luís, MA. Do total de 60 amostras analisadas, 96,6 por cento apresentaram-se contaminadas. Foram identificados ovos de ancilostomídeos, Ascaris lumbricoides, Enterobius vermiculares, Hymenolepis sp., Taenia sp., oocistos de coccídeo, cistos de Entamoeba sp. Também se detectou ácaros, ovos de ácaro, fragmentos de ácaros e artrópode, piolho, hifas de fungo, larvas de nematóides. Conclui-se que, as hortaliças já saem das hortas contaminadas e podem veicular enteroparasitas para a população.