RESUMEN
BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.
Asunto(s)
Doxorrubicina/administración & dosificación , Melanoma Experimental/diagnóstico por imagen , Melanoma Experimental/tratamiento farmacológico , Ácidos Polimetacrílicos/administración & dosificación , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/tratamiento farmacológico , Administración Intravenosa , Animales , Carbocianinas/química , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Humanos , Macrófagos/química , Melanoma Experimental/química , Melanoma Experimental/patología , Ratones , Microscopía Electrónica , Nanopartículas , Trasplante de Neoplasias , Tubo Neural/química , Neutrófilos/química , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Neoplasias Cutáneas/química , Neoplasias Cutáneas/patología , Resultado del Tratamiento , Pez CebraRESUMEN
We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line (1-3) and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP (4). In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.
Asunto(s)
Células Neuroepiteliales/citología , Análisis de la Célula Individual/métodos , Animales , División Celular/fisiología , Línea Celular , Femenino , Colorantes Fluorescentes/química , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Tubo Neural/química , Tubo Neural/citología , Tubo Neural/embriología , Células Neuroepiteliales/metabolismo , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
As pharmacists and pharmacy students are increasingly called upon to assume roles in public health activities, it is important to recognize unique opportunities to educate community members on health, wellness, and disease prevention. Objective: To evaluate the impact of a pilot health promotion program on college women's knowledge regarding folic acid and prevention of neural tube defects (NTD) and frequency of multivitamin use. Methods: A health promotion program was developed by a pharmacy student and two pharmacy faculty members that included an oral presentation and reminder messages. A multiple-choice test assessing knowledge of folic acid and NTD and frequency of multivitamin use was given to participants before and immediately after the presentation. Participants then received a reminder message regarding folic acid once a week for three weeks. Knowledge and multivitamin use were re-assessed four weeks post-intervention. Results: Thirty-two college women voluntarily attended the oral presentation. Twenty-five women (78.2%) completed the four-week post-test. Compared to the pre-test, there were statistically significant increases in average test score (p<0.0001) and correct responses to questions regarding folic acid and NTD (p<0.05 for each question). Participants reported a statistically significant increase in regular (> 4 times/week) multivitamin use (p=0.023). Conclusion: Participants in the pilot health promotion program demonstrated a statistically significant increase in knowledge about folic acid and frequency of multivitamin use. A similarly-modeled health promotion program may be an effective way of increasing folic acid and NTD knowledge and changing behaviors of multivitamin use in college women (AU)
Como los farmacéuticos y estudiantes de farmacia están llamados cada día más a asumir papeles en actividades de salud pública, es importante reconocer las oportunidades para educar a los miembros de la comunidad en salud, bienestar y prevención de la enfermedad. Objetivo: Evaluar el impacto de un programa piloto de promoción de la salud en el conocimiento de las universitarias sobre ácido fólico y prevención de defectos del tubo neural (DTN) y la frecuencia del uso de multivitamínicos. Métodos: Se desarrolló un programa de promoción de la salud por un estudiante de farmacia y dos profesores de la facultad que incluía una presentación oral y mensajes recordatorios. Se dio a las participantes antes e inmediatamente después de la presentación un test de respuesta múltiple que evaluaba el conocimiento sobre ácido fólico y DTN y la frecuencia de uso de multivitamínicos. Las participantes recibieron después un mensaje recordatorio sobre el ácido fólico una vez a la semana durante tres semanas. Los conocimientos y el uso de multivitaminicos fueron re-evaluados cuatro semanas después de la intervención. Resultados: 32 universitarias asistieron voluntariamente a la presentación oral. 25 mujeres (78,2%) completaron las cuatro semanas post-intervención. Comparado con el pre-test, hubo incrementos estadísticamente significativos en la puntuación media del test (p<0,0001) y en las respuestas correctas a preguntas sobre ácido fólico y DTN (p<0,05 para cada pregunta). Las participantes comunicaron un aumento estadísticamente significativo en el uso regular (> 4 veces/semana) de multivitamínicos (p=0,023). Conclusión: Las participantes en el programa piloto de promoción de la salud demostraron un aumento estadísticamente significativo en conocimientos sobre el ácido fólico y la frecuencia de uso de multivitamínicos. Programas diseñados de modo similar podrían ser efectivos para aumentar el conocimiento sobre ácido fólico y DTN y cambiar las actitudes sobre el uso de complejos vitamínicos entre universitarias (AU)
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Estudiantes de Farmacia/estadística & datos numéricos , Educación en Farmacia/normas , Conocimientos, Actitudes y Práctica en Salud , Promoción de la Salud/métodos , Promoción de la Salud , Ácido Fólico/farmacología , Ácido Fólico/uso terapéutico , Tubo Neural/anomalías , Tubo Neural/química , Tubo Neural , Salud Pública/métodos , Proyectos Piloto , Vitaminas/uso terapéutico , Estados Unidos/epidemiologíaRESUMEN
Neural tube defects (NTDs), such as spina bifida, are common and severe birth defects in humans but the underlying causes are poorly understood. The pathogenesis and etiology of spina bifida in the curly tail mouse closely resemble defects in humans, providing a well-characterized model of NTDs. Grainyhead-like-3 (Grhl3), which encodes a transcription factor, was recently identified as a candidate gene for curly tail based on chromosomal location and the occurrence of spina bifida in Grhl3 null mice. However, the causative curly tail mutation has not been established, while the relationship between Grhl3 gene expression and the known cellular defect leading to NTDs in curly tail is unknown. Spina bifida in curly tail results from a cell type-specific proliferation defect in the hindgut endoderm, and we find that Grhl3 is expressed specifically in this tissue during the final stages of spinal neural tube closure in wild type embryos. Moreover, Grhl3 expression is diminished in the spinal region of neurulation-stage curly tail embryos. Curly tail mice do not carry a coding region mutation in Grhl3, however, we found a putative regulatory mutation upstream of the Grhl3 gene, which may be responsible for the expression deficit. In order to test the hypothesis that spina bifida in curly tail mice results from insufficient expression of Grhl3, we generated Grhl3-expressing curly tail mice by bacterial artificial chromosome-mediated transgenesis and demonstrated complete rescue of spina bifida. This study provides evidence for a critical role of diminished Grhl3 expression in causing spinal NTDs in the curly tail mouse model.