Immunophenotyping of progenitor cells from articular cartilage of New Zealand Rabbits (Oryctolagus cuniculus).

Mesenchymal stem cell (MSC) have immunomodulatory and anti-inflammatory effects, allowing its application in the therapy of different diseases, including articular cartilage injuries, which induce the establishment of a pro-regenerative microenvironment in the injured tissue. Therefore, our objective was to isolate, characterize and differentiate cartilage cells from different joints of New Zealand rabbit (Oryctolagus cuniculus), in order to verify their potential as MSC for future clinical use. For this, cartilage fragments were isolated from the humerus-radio-ulnar joints, humeral scapula, femoro-tibio-patellar, and lame femoris from rabbits. The results showed that the cells were rounded in the center of the plate and fibroblastoids in the periphery. After thawing, the cells did not change their growth time in culture, nor their morphology. The cells showed labeling for mesenchymal stem cell, cytoskeleton, pluripotency and cell proliferation, but not for hematopoiesis markers (CD105+ and CD34-). We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. After application of these cells in nude mice, no tumor growth was observed in spleen, kidney, liver, lung and heart. Therefore, we conclude that cells isolated from the articular cartilage of rabbits present characteristics of MSC with potential for future clinical applications.

Gonadal differentiation during embryonic and fetal development of male New Zealand rabbits (Oryctolagus cuniculus) / Diferenciación gonadal durante el desarrollo embrionario y fetal de conejos machos neozelandeses (Oryctolagus cuniculus)

SUMMARY: The rabbit is considered an ideal animal model for studies that describe abnormalities in the testicles due to the similar morphogenetic mechanisms of sexual development and diseases commonly found in humans. The aim of this study was to determine the male sexual differentiation of the New Zealand rabbit (Oryctolagus cuniculus) through development. The gestational age was estimated and classified as 9, 12, 14, 16, 18, 20, 23 and 28 gestational days. The morphological and sexual determination were performed by histological analysis of the reproductive tract in the embryos and fetuses (9-28 days) as well as by immunohistochemistry- Desert hedgehog-Dhh- (testis-specific protein on Y chromosome- 16, 20, 23 days and adult rabbits). Gonads were observed from the 14th day in an undifferentiated stage and with homogeneous aspect. Sexual differentiation was observed from the 16th day with presence of cells forming gonadal cords and Dhh+ cells in the gonadal parenchyma. From the 18th gestational day testicular cords were observed, which evolved into organized seminiferous tubules. The formation of the efferent ducts and ductus deferens and epididymis was observed on the 20th and 23rd days, respectively. The differentiation of the external genitalia occurred from the 23rd days from the anogenital distance and was identified to identify the penile structures. In summary, the features of the sexual differentiation were determined by observation of the Dhh+ protein in embryos from the 16th day to adulthood, and the morphological particularities observed from the 18th gestational day, determined by differentiation of the external genitalia from the 23rd day.

RESUMEN: El conejo se considera un modelo animal ideal para estudios que describen anomalías a nivel testícular debido a que presenta mecanismos morfogenéticos similares al desa- rrollo sexual y enfermedades que se encuentran comúnmente en los seres humanos. El objetivo de este estudio fue determinar la diferenciación sexual masculina del conejo de Nueva Zelanda (Oryctolagus cuniculus) a través del desarrollo. La edad gestacional se estimó y clasificó en 9, 12, 14, 16, 18, 20, 23 y 28 días gestacionales. La determinación morfológica y sexual se realizó mediante análisis histológico del tracto reproductivo en los embriones y fetos (9 - 28 días) así como mediante inmunohistoquímica -Desert hedgehog-Dhh- (proteína testicular específica en el cromosoma Y- 16, 20, 23 días y conejos adultos). Las gónadas se observaron a partir del día 14 en un estadio indiferenciado y con aspecto homogéneo. Se observó diferenciación sexual a partir del día 16 con presencia de células formadoras de cordones gonadales y células Dhh+ en el parénquima gonadal. A partir del día 18 de gestación se observaron cordones testiculares, que evolucionaron a túbulos seminíferos organizados. La formación de los conductos eferentes, deferentes y del epidídimo se observó a los 20 y 23 días, respectivamente. La diferenciación de los genitales externos ocurrió a partir del día 23 desde la distancia anogenital y se utilizó para identificar las estructuras del pene. En conclusión, las características de la diferenciación sexual se determinaron mediante la observación de la proteína Dhh en embriones desde el día 16 hasta la edad adulta, y las particularidades morfológicas observadas a partir del día 18 de gestación, determinadas por diferenciación de los genitales externos a partir del día 23.

Stereological analysis of metanephros from domestic cat (Felis catus, Linnaeus 1798) embryos and fetus.

Kidney diseases are the most common illness for cats with a prevalence seven times higher than in dogs. Metanephros is the last of three renal systems to be formed during the embryonic period, which then becomes the permanent kidney. The current work aimed to analyse the morphology and to quantify the structures present in the development of metanephros from domestic cat (Felis catus) embryos and foetuses. For this purpose, the evaluation of the biometric parameters of metanephros from cat embryos and foetuses was performed in addition to the quantification of renal corpuscles and volume of cortical and medullary layers by stereological analysis. The evaluated biometric parameters were weight, width, height, thickness and volume. The values of the measured biometric parameters increased throughout the gestational stages. The quantity of renal corpuscles gradually increased following the embryo-foetal development, mainly during the middle of the gestational stage. It was during this phase that morphologically, a complete corticomedullary division was observed. Although the difference in the quantity of renal corpuscles between the middle and the end of the gestational stages was not statistically significant, there was an increase in the volume of the medullary layer and a decrease in the volume of the cortical layer between these two stages. These findings suggest that the metanephros presents a progressive growth with the renal corpuscles following this development until the middle of the gestational stage. Starting from this phase, the differentiation of the corticomedullary layers can be seen with a significant increase in the medullary layer.

Evaluation of immunohistopathological profile of tubular and solid canine mammary carcinomas.

Breast cancer is the most common cancer in women, but the incidence of mammary carcinoma in female dogs is even higher than in humans. These two tumors have similarities that can be seen by its biological behavior, molecular genetic alterations, and histology. This suggest that female dogs can be an excellent model for preclinical oncological studies. And the mammary carcinoma most frequently found in this species is the tubular and solid carcinomas. The extracellular matrix (ECM) has an important role in the progression of these tumors. Because of that we proposed to evaluate the ECM components of these carcinomas through histology with specific stains such as Masson's Trichrome, Picrosirius Red and the technique of scanning electron microscopy. With that, we found the presence of collagen fibers in the tubular carcinoma and around its parenchyma. On the other hand, the solid carcinoma presented collagen fibers throughout the parenchyma and around each tumor cell. With the transmission electron microscopy, we observed the presence of mitochondrias and rough endoplasmic reticulum in both tumors. And finally, we evaluated the expression of proteins through the immunohistochemistry, in which we found a high expression of VEGF, PCNA, CK-18 and vimentin in solid carcinoma, and a positive mark in the tubular and solid carcinoma for collagen I, III and fibronectin. Thus, we demonstrated some differences in the ECM of these mammary carcinomas, allowing a better understanding of its histological characteristics, and these data may contribute to future studies about therapies focused on tumors ECM.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation.

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin-eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.

Canine amniotic membrane mesenchymal stromal/stem cells: Isolation, characterization and differentiation.

The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tumorigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the immunological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications.

Morphology of the Oligoryzomys nigripes respiratory system.

Rodents are important in the transmission of infectious diseases that affect the respiratory tract, including simple infections and those caused by specific pathogens. These animals are natural reservoirs of zoonoses that cause many public health diseases. Basic knowledge on the morphology of these animals is important as basic research is useful for applied studies, such as the development of clinical, therapeutic, surgical and clinical models. Morphological data of respiratory tract in Oligoryzomys nigripes are absent in the literature. Therefore, the aim of this study was to perform a morphological analysis of the respiratory tract of O. nigripes. Five adult females from the environmental reserve in São Joaquim da Barra, São Paulo were used, donated to the Museum of Veterinary Anatomy (FMVZ/USP). Several morphological features follow the same pattern seen in rodents; however, this species showed some differences such as the presence of three lobar bronchi, nonlobed left lung and the right lung constituted by two lobes. Respiratory epithelium lined the whole respiratory tract and was seen using scanning electron microscopy the oval shape of the parenchyma and alveoli.

Sexual determination and differentiation during embryonic and fetal development of New Zealand rabbit females / Determinación y diferenciación sexual durante el desarrollo embrionario y fetal de conejos hembras de Nueva Zelanda

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.

El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.

Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells.

BACKGROUND: Stem cells are capable of unlimited self-renewal and are able to remain undifferentiated for extended periods of time prior to their differentiation into specific cell lineages. Because of the issues (ethical and religious) involved in the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative cell source could be foetal stem cells derived from extra-embryonic tissue, which are highly proliferative, grow in vitro and possess interesting immunogenic characteristics. As a result, the amniotic membrane of several species has been studied as an important new source of stem cells. METHODS: Here, we cultured and characterized mesenchymal progenitor cells derived from the rabbit amniotic membrane, and investigated their differentiation potential. In total, amniotic membranes were collected from eight rabbit foetuses and were isolated by the explant technique. The obtained cells were cultured in DMEM-HIGH glucose and incubated at 37 °C in a humidified atmosphere with 5% CO2. RESULTS: The cells adhered to the culture plates and showed a high proliferative capacity with fibroblast-like morphologies. The cells showed a positive response for markers for the cytoskeleton, mesenchymal stem cells and proliferation, pluripotency and haematopoietic precursor stem cells. However, the cells were negative for CD45, a marker of haematopoietic cells. Furthermore, the cells had the capacity to be induced to differentiate into osteogenic, adipogenic and chondrogenic lineages. In addition, when the cells were injected into nude mice, we did not observe the formation of tumours. CONCLUSIONS: In summary, our results demonstrate that multipotent mesenchymal stem cells can be obtained from the rabbit amniotic membrane for possible use in future cell therapy applications.

Cochlear epithelial of dog fetuses: a new source of multipotent stem cells.

Hearing loss caused by the damage of cochlea sensory cells or neurons is a common human disease, but also affects dogs and other animals. To test their progenitor nature as potential value for future therapies, we characterized cells derived from the cochlear epithelium in dog fetuses. In total, 8 fetuses of 35-40 days of gestation, derived from castration campaigns, were investigated. Cells were analysed by the MTT colorimetric assay and in regard to cell cycle, differentiation capacities, immunophenotypes and qPCR analysis. In culture, cells had a fibroblast-like morphology. Phenotypic immunocharacterization showed positive staining for mesenchymal stem cell and pluripotency markers and were negative for hematopoietic cell markers. Cells possessed differentiation capacity for the three main cell lineages: osteogenic, adipogenic and chondrogenic, altogether indicating their nature as mesenchymal stem cells. Thus, cells derived from fetal cochlear tissues indeed may provide valuable sources of progenitor cells for cell therapy of canine deafness and other diseases.