Morphological and ultrastructural characterization of neurospheres spontaneously generated in the culture from sheep ovarian cortical cells.

Neurospheres (NS) derived from adult stem cells of non-neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy. Knowing the fine structure of NS cells is essential for characterizing them during differentiation or oncogenic transformation. NS are generated by culturing ovarian cortical cells (OCCs) under specific conditions. To establish whether these OCCs exhibited a similar morphophenotype as those from the central nervous system (CNS) reported in the literature, sheep OCCs were cultured for 21 days to generate NS. Expression levels of pluripotency (Nanog, octamer-binding transcription factor 4 [Oct4], and SRY-box transcription factor 2 [Sox2]) and NSCs/NPCs (nestin, paired box 6 [Pax6], and p75 neurotrophin receptor [P75NTR]) transcripts were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the NSC/NPC antigens were immunolocalized, and structural and ultrastructural analyses were performed in OCC-NS on Days 10, 15, and 21 in culture. Spheroids expressed transcripts and antigens of pluripotency as well as NSCs/NPCs. Cells were arranged into an inner core, with frequent apoptotic and degenerative events, and a peripheral epithelial-like cover with abundant cytoplasmic organelles, apical microvilli, and filament bundles of cytoskeleton elements. Adherens junctions and apical tight and lateral loose interdigitations were found in peripheral cells that eventually lost apical-basal polarization, which might indicate their disengaging/aggregating from/to the NS. We can conclude that OCC-NS shares the most structural and ultrastructural characteristics with CNS-NS.

Spheroids Spontaneously Generated In Vitro from Sheep Ovarian Cortical Cells Contain Integrating Cells That Exhibit Hallmarks of Neural Stem/Progenitor Cells.

Cell spheroids are inducible or spontaneously generated cell aggregates produced in vitro that can provide a valuable model for developmental biology, stem cell biology, and cancer therapy research. This investigation aimed to define the cellular identity of spheroids spontaneously generated in vitro from sheep ovarian cortical cells cultured under specific serum-free conditions. Spheroids were characterized during 21 days of culture by morphometric evaluation, detection of alkaline phosphatase (AP) activity, gene expression analyses of stemness transcription factors and several lineage markers, immunolocalization analyses, as well as assessment of self-renewal and differentiation potential. Cell aggregation, evidenced from day 3 of culture onward, resulted in efficient generation of 65-75 spheroids for every 500,000 cells seeded. The spheroids reached maximum diameter (187 ± 15.9 µm) during the second week of culture and exhibited AP activity. Sox2, Oct4, and Nanog were expressed throughout the culture period, with upregulation of Sox2. Neural lineage specification genes (eg, nestin, vimentin, Pax6, and p75NTR) were expressed from day 10 onward at levels above that of Oct4, Nanog and those for endoderm [alpha-fetoprotein (AFP)], and mesoderm (brachyury) specification. Neural stem cell (NSC)/neural progenitor cell (NPC) markers, nestin, Pax6, p75NTR, and vimentin, were extensively localized in cells on day 10, 15 (44.75% ± 5.84%; 93.54% ± 1.35%; 78.90% ± 4.80%; 73.82% ± 3.40%, respectively), and 21 (49.98% ± 5.30%; 91.84% ± 1.9%; 76.74% ± 11.0%; 95.80% ± 3.60%, respectively). Spheroid cell self-renewal was evidenced by cell proliferation and the generation of new spheroids during two consecutive expansion periods. Culture of spheroid cells under differentiation conditions gave rise to cells showing immunolocalization of the neuron-specific antigen NeuN and the astroglial antigen GFAP (glial fibrillary acidic protein). Our results indicate that spheroids spontaneously generated in this culture system were comprised of cells with molecular characteristics of NSC/NPC that can self-renew and differentiate into neurons and glia, supporting the identity of spheroids as neurospheres.

Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer.

BACKGROUND: Indole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer. METHODS: The aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks. RESULTS: Indole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum. CONCLUSIONS: Our data reveal for the first time that the ingestion of indole-3-carbinol, as administered, diminishes proliferation and increases apoptosis of tumor cells in an experimental model of inflammatory breast cancer, although this effect could not be enough to avoid the appearance of tumor embolization and metastasis. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved.

Corticoadrenal and Cardiorespiratory Responses to Administration of Propofol Combined with Dexmedetomidine or Ketamine in Rabbits.

Anesthetic protocols may influence adrenal function. Effective methods for modulating stress are desirable to minimize secondary effects during the perioperative period. The aim of this study was to evaluate the effects of the administration of propofol with dexmedetomidine or ketamine on corticoadrenal function and heart and respiratory rates. A random treatment-order design was used: each rabbit received all treatments, with at least 14 d between experiments. Rabbits were assigned to 3 treatment groups (10 per group): group 1, 1 mL normal saline solution intravenously; group 2, propofol (3 mg/kg IV) and dexmedetomidine (0.35 mg/kg IM); and group 3, propofol (3 mg/kg IV) and ketamine (1 mg/kg IV). Dexmedetomidine was injected 15 min prior to propofol administration. Blood samples were obtained before drug administration and at 5, 10, 30, and 60 min and 24 h after injection. Serum cortisol and corticosterone levels were measured by competitive enzyme immunoassay. Serum glucocorticoid concentrations did not change in group 2. However, rabbits in group 3 showed an increase in serum cortisol (at 5-60 min) and corticosterone (at 5-120 min) when compared with all other groups at the corresponding time points. This increase probably reflected both propofol- and ketamine-associated stimulatory effects corticoadrenal function. Respiratory rate decreased in groups 2 and 3 animals, and heart rate decreased in group 2, probably due to sympathetic inhibition by propofol and dexmedetomidine. In conclusion, propofol-ketamine provides suitable cardiorespiratory stability in rabbits but enhances glucocorticoid secretion more than dexmedetomidine-propofol anesthesia. Glucocorticoid levels in anesthetized rabbits should be considered during protocol design to minimize the stress response to surgery and to avoid erroneous data interpretation.

Effects of Dexmedetomidine and Ketamine-Dexmedetomidine with and without Buprenorphine on Corticoadrenal Function in Rabbits.

Anesthetics may influence adrenal function and consequently alter serum glucocorticoid concentrations, leading to erroneous interpretations of results from anesthetized rabbits. However, decreases in glucocorticoid concentrations may be advantageous in protocols designed to minimize the stress response to surgery. This study characterized the variations in adrenocortical function based on changes in corticosterone and cortisol levels after various doses and combinations of dexmedetomidine, ketamine, and buprenorphine. Each rabbit received all treatments with a minimal interexperiment interval of 10 d. Rabbits were allocated to 7 groups (n = 10 per group) and received either 1 mL saline solution; dexmedetomidine at 0.05, 0.15, or 0.25 mg/kg; ketamine (35 mg/kg) and dexmedetomidine (0.25 mg/kg) without or with buprenorphine (0.03 mg/kg); or ketamine (35 mg/kg) and buprenorphine (0.03 mg/kg). Blood was sampled before drug administration and at 10, 30, 60, and 120 min and 24 h afterward. Serum glucocorticoid levels fell in all treatment groups except the one receiving ketamine-dexmedetomidine; in that group, serum glucocorticoids increased. Rabbits that received ketamine-dexmedetomidine-buprenorphine had the lowest serum glucocorticoid levels overall. In conclusion, dexmedetomidine reduces glucocorticoid secretion in rabbits but, when combined with ketamine, increases corticosterone and cortisol levels as well as heart and respiratory rates. The addition of buprenorphine to the ketamine-dexmedetomidine mixture reduces serum glucocorticoid levels. The influence of anesthetic drugs should be considered when designing a protocol to minimize the glucocorticoid response to surgery or when measuring glucocorticoid levels in rabbits.

Stress physiological responses to tourist pressure in a wild population of European pine marten.

The tourist pressure in natural parks is a potential source of stress and may cause an increase in the adrenal activity of wild populations of European pine marten (Martes martes). Seventy-six faecal samples were collected during 15 months in a natural park of Northwest Spain. Analysis of faecal DNA was used for the specific identification using the PCR-RFLPs technique. Faecal steroid determinations were performed by EIA. Natural park was divided in three areas: free entry, restricted area, and integral reservation, and number of daily human visitors recorded. Faecal glucocorticoid metabolite levels (ng/g dry faeces) were significantly higher in spring (56.36+/-19.62) and summer (31.27+/-11.98) compared to autumn (15.33+/-6.89) and winter (11.13+/-3.30). These data are closely related to daily number of visitors (spring: 3204, summer: 1672, winter: 646, autumn: 551). Androgen, progestin and oestrogen levels were also significantly higher in spring (reproductive season) showing values of 43.62+/-18.6, 154.31+/-53.50 and 829.62+/-456.1, respectively. Glucocorticoid levels were significantly lower in integral reservation (15.95+/-3.56) compared to restricted (31.4+/-16.30) and free entry areas (41.59+/-12.73), respectively. Wild populations of European pine marten showed stress physiological response induced by the tourist pressure and this response is higher during reproductive season.