Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

1. Entrega hidrodinámico de ADN MC sRANKL <ol><li> Entrega hidrodinámica a través del ratón de la cola de la vena <ol><li> Pesar el ratón antes de la inyección en la vena de la cola. Diluir sRANKL o proteína fluorescente verde (GFP) MC en solución de Ringer (pre-calentamiento a 37 ° C) en un volumen total de ~ 10% del peso corporal del ratón. </li><li> Caliente el ratón en una jaula durante 10 min antes de la inyección con el fin de dilatar los vasos sanguíneos y hacer que las venas laterales (LVs) visibles. Monitorear el ratón con cuidado para evitar la deshidratación y la hipertermia. </li><li> Tan pronto como los volúmenes lógicos están dilatados y visibles, transferir el ratón a la inmovilización e inserte el conector lo suficiente en el barril para restringir el movimiento. Monitorear el ratón para la actividad normal, como la respiración. Ajuste el tapón de inmovilización, si es necesario. </li><li> Desinfectar el área de la inyección de 3/4 de la punta de la cola con un algodón con alcohol. Utilice una aguja de 27 a 30 para el ratón inyección en la vena de la cola. Sostenga la cola firmemente con una mano e inserte la aguja ena la vena de la cola. Aplique presión sobre la jeringa y completar la inyección dentro de 5-7 seg. </li><li> Retire la aguja de la vena de la cola y aplique presión con un algodón sobre el lugar de la inyección para detener el sangrado. Monitorear el ratón durante 15-30 minutos, a continuación, transferir el ratón de nuevo a vivarium una vez que el ritmo de la respiración se reduce a su ritmo normal. </li></ol></li><li> Confirmación de la expresión y cuantificación de Ratón Serum TRACP5b Mensaje Gene Transfer sistémica Ratón sRANKL <ol><li> Caliente el ratón en una jaula durante 5-10 minutos con el fin de dilatar los vasos sanguíneos y hacer LV visible. Monitorear el ratón con cuidado para evitar la deshidratación y la hipertermia. </li><li> Hacer una pequeña incisión con una cuchilla a la cola del ratón en un sitio de una cuarta parte de la punta de la cola. </li><li> Recoge alrededor de 100 l de sangre en tubos de separación de suero. </li><li> Incubar los tubos de separación de suero a temperatura ambiente durante 30 min. </li><li> Centrifugar las muestras a 10.000 xg durante 5 min y recoger el suero. Store suero en -80 ° C para su posterior análisis. </li><li> Realizar sRANKL ELISA y ensayo de TRACP5b suero de ratón en muestras de suero utilizando un kit comercial disponible. </li></ol></li></ol> 2. In vitro generación de osteoclastos de Ratón BMMs <ol><li> Aislamiento y cultivo de BMMs <ol><li> El aislamiento de la tibia y los huesos de fémur: La eutanasia el ratón donante con CO 2, y desinfectar las piernas con etanol al 70%. Diseccionar desde el pubis hasta el hueso calcáneo, para quitar los huesos fémur y la tibia intacta. </li><li> Quite la piel y el músculo cuidadosamente sin dañar el hueso. Coloque fémur procesado y hueso de la tibia en una placa de Petri que contiene tampón fosfato salino (PBS). </li><li> La eliminación de la médula ósea: Corte la punta en un lado de los huesos del fémur y de la tibia y enjuagar la médula ósea en un tubo de 50 ml utilizando una jeringa de 1 ml con una aguja de calibre 25, cargado con α-MEM que contiene 1% de penicilina / estreptomicina (osteoclastos Cultura Medio / OCM). </li><li&gt; Proceso de médula ósea: Mezclar y transferir la suspensión de la médula ósea a un nuevo tubo de 50 ml pasando a través de un filtro de células de 70 m de nylon. Centrifugar las células a 300 xg y volver a suspender en α-MEM que contiene 1% de penicilina / estreptomicina y 10% de FBS (medio de osteoclastos Cultura / OCM (+)). </li></ol></li><li> Cultura de los macrófagos <ol><li> Contar las células utilizando un hemocitómetro o un contador automático de células. </li><li> Plate 1 x 10 6 células por pocillo en una placa de 6 pocillos o 3 x 10 5 células a cubreobjetos de vidrio (5 mm) o rodajas de dentina colocados en placas de 96 pocillos y se incuba a 37 ° C. </li><li> Aspirar todos los medios que contienen las células no adherentes y se incuban las células a 37 ° C con OCM fresca (+) que contiene 25 ng / ml ratón M-CSF durante 48 horas. </li><li> Aspirar todos los medios de comunicación y se incuban las células a 37 ° C con frescos OCM (+) un medio que contenía 25 ng / ml ratón M-CSF, y 30 ng / ml sRANKL ratón para iniciar la osteoclastogénesis. Reponer los medios de comunicación cada 3 días según sea necesario. </li><li> Se cultivan las célulasdurante aproximadamente 6-8 días para formar células multinucleares gigantes capaces de resorción ósea. En cuanto aparezcan células multinucleadas, realice TRAP (fosfatasa ácida resistente al tartrato) tinción utilizando un kit comercial disponible, y cuando está completamente madura, realizar ensayos funcionales. Visualizar anillos de actina F en cubreobjetos utilizando mancha faloidina y la imagen de las células unidas a la dentina rebanadas por microscopía electrónica de barrido (SEM) como se ha descrito previamente 19. </li></ol></li></ol> 3. Generación de osteoclastos in vitro de las PBMC humanas <ol><li> Aislamiento de PBMC humanas <ol><li> Añadir 10 ml de pre-calentado Histopaque-1077 en un tubo de 50 ml. </li><li> Filtro de leucocitos vacío obtiene a partir del banco de sangre con PBS estéril en un tubo de 50 ml (proporción de 1:1 de sangre con PBS). Capa de la sangre diluida sobre la solución Histopaque muy lentamente, asegurándose de que la sangre no se mezcla con el histopaque. </li><li> Centrifugar las muestras a 1000 xg durante 20 min a 18 ° C. Seguiring centrifugación, aislar cuidadosamente la capa de la capa leucocitaria blanca que contiene las células blancas de la sangre y transferir a un nuevo tubo de 50 ml. </li><li> Diluir las células con PBS y se centrifuga de nuevo a 650 xg durante 5 minutos para recoger las células. </li><li> Aspirar el sobrenadante y volver a suspender las células en OCM (-) de los medios y contar las células utilizando un contador de células. </li></ol></li><li> Plating y cultivo de CMSP <ol><li> Resuspender las células en las OCM (+) los medios de comunicación y la placa de 8 x 10 6 células / ml por pocillo en una placa de 6 pocillos o 1 x 10 6 células / ml por pocillo en placas de 96 pocillos. Las células de la placa sobre cubreobjetos de vidrio (5 mm) según se requiera para obtener imágenes de inmunofluorescencia, así como en secciones de hueso adecuados para ensayos de resorción ósea como anteriormente descritos 20. </li><li> Cambio de los medios de cultivo, la reposición de las células con M-CSF humano cada 2-3 días o según sea necesario. A continuación, proceder a la etapa de diferenciación en el día 5 por la adición de 30 ng / ml de sRANKL humana junto con 25 ml de M-CSF ng / humano en OCM (+) medios para iniciar osteoclastogeneses. </li><li> Se cultivan las células durante aproximadamente 14 a 21 días para formar células multinucleares gigantes. Tan pronto como aparecen células multinucleadas, a continuación, estas células se pueden etiquetar para TRAP, y cuando está completamente madurado, ensayos funcionales pueden llevarse a cabo. Anillos de actina F se pueden visualizar en cubreobjetos utilizando tinción phalloidin. La morfología de las células puede examinarse en células unidas a la dentina rebanadas por SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Las afecciones musculoesqueléticas son las principales causas de morbilidad y discapacidad, y se componen de más de 150 enfermedades y síndromes; que afecta hoy a unos 90 millones de estadounidenses. Inflamación de las articulaciones y la destrucción ósea son las características predominantes de los trastornos musculoesqueléticos, como la artritis y la osteoporosis. La osteoporosis es una enfermedad que debilita la integridad ósea, a menudo conduce a las fracturas de los huesos. La artritis es una enfermedad cr...

Enfermedades musculoesqueléticas afectan a millones de personas en los Estados Unidos y del presente graves consecuencias para los sistemas de salud nacionales y locales 1. Estos trastornos se caracterizan por la pérdida de hueso y función de las articulaciones que requieren tratamiento extenso y largos períodos de recuperación. Comúnmente, un aumento relativo en el número y / o actividad de los osteoclastos, células especializadas para reabsorber el hueso, en la osteoporosis y la artritis se observa 2. En condiciones fisiológicas el número y la actividad de los osteoclastos está regulada por el activador del receptor del factor nuclear κ-B ligando (RANKL), que es producida por los osteoblastos. La osteoprotegerina (OPG), un receptor señuelo para RANKL también es producida por los osteoblastos 3 modelos animales in vivo que implican la sobreexpresión sistémica de sRANKL, o supresión de OPG son muy valiosa en la investigación de la osteoporosis.; Sin embargo, estos métodos requieren la generación de ratones transgénicos 4,5. Aquí, una nueva alternativamétodo de sobreexpresar sRANKL para el estudio de los trastornos musculoesqueléticos relacionados-se describe. Específicamente, minicircle (MC) la tecnología del ADN y los métodos de entrega hidrodinámicas se utilizaron para lograr la transferencia de genes de sRANKL in vivo e sobreexpresan ratón sRANKL sistémicamente 6. Este método también es complementaria a otros modelos in vivo de la osteoporosis, tales como la modulación hormonal de los osteoclastos siguientes ovariectomía 7 y la intervención dietética con dieta baja en calcio 8. Estos modelos son muy útiles para estudiar diferentes aspectos de los trastornos musculoesqueléticos relacionados-sin embargo, requieren de procedimientos quirúrgicos y pueden durar hasta varios meses, a un costo significativo 9. Ovariectomizadas (OVX) modelo de roedores es un modelo animal experimental en que la extirpación de los ovarios conduce a la deficiencia de estrógenos imitando así la osteoporosis posmenopáusica humana 10. La osteoporosis post-menopáusica humana, una condición donde Defici estrógenoparencia conduce a un mayor riesgo de fracturas óseas y la osteoporosis afecta a aproximadamente ocho millones de mujeres en los Estados Unidos solamente. Aunque el modelo OVX es útil para la osteoporosis post-menopáusica que ofrece ventajas limitadas en el estudio de la osteoporosis en general. El estrógeno suprime la pérdida de hueso, mediante la inducción de osteoclastos y la inhibición de la apoptosis de los osteoblastos, por lo tanto, en su ausencia se observa un aumento de la actividad de los osteoclastos 10-12. Un desequilibrio en la relación RANKL-OPG que favorece la resorción ósea también se observa 13. Sin embargo, la deficiencia de estrógenos en vivo también se acompaña de disminución de los niveles de transformación del factor de crecimiento β (TGF β), aumento de la interleucina-7 (IL-7) y de TNF, IL-1 e IL-6 14,15. Como estas citoquinas han conocido funciones moduladoras de remodelación ósea independiente de la vía de RANKL, es imposible atribuir cualquier activación de los osteoclastos exclusivamente al eje RANKL-RANK. El modelo que se describe en este documento permite a los investigadores estudiar en vivo eje RANKL a RANK en la osteoclastogénesis y la pérdida ósea sin las citoquinas pro-inflamatorias en comparación con modelos de roedores OVX. Además, las técnicas de la osteoclastogénesis in vitro son herramientas esenciales para el estudio de la activación de los osteoclastos para posibles tratamientos terapéuticos de las enfermedades musculoesqueléticas. Estudios anteriores también han demostrado que el cultivo de macrófagos derivados de médula ósea de ratón (BMMs) con macrófagos de ratón factor estimulante de colonias (M-CSF) y el ratón sRANKL puede conducir a la diferenciación de los osteoclastos 3,16,17. Aquí se describen los protocolos para generar células similares a osteoclastos multinucleadas de médula ósea de ratón, así como de células de sangre periférica humana (PBMC) in vitro 18. Los ensayos basados ​​en células necesarios para definir un osteoclastos terminales diferenciadas y completamente funcional madura también se describen brevemente. Estas técnicas in vitro complementan la novela en el enfoque vivo y juntos sirven como pherramientas de investigación para estudiar owerful diferenciación de los osteoclastos y la activación. El uso de estos sistemas, los científicos son capaces de generar los osteoclastos in vivo e in vitro y definir los estímulos y señales requeridas para su proliferación y activación, así como probar la eficacia de los inhibidores farmacológicos y biológicos.

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a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Aquí, una técnica de transferencia de gen nuevo para la diferenciación de los osteoclastos in vivo y protocolos de cultivo de células para la diferenciación de células precursoras en los osteoclastos in vitro como un método para estudiar los efectos de las citoquinas en la osteoclastogénesis se describen. En la Figura 1, se muestran los resultados representativos de la transferencia de genes exitosa de GFP y ratón sRANKL MC en ratones. En la Figura 2, se muest...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica de transferencia de genes y<em&gt; In vitro</em&gt; ensayos basados ​​en células para el estudio de la pérdida ósea en los trastornos musculoesqueléticos

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K vistas.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Una estrategia para identificar mutaciones de novo en los trastornos comunes, tales como el autismo y la esquizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Investigación

Medicina

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Establecimiento y propagación de los tumores del retinoblastoma humanos en ratones con deficiencia inmune

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Evaluación longitudinal de ratón posteriores pérdida de masa ósea del miembro después de una lesión de la médula espinal utilizando Novel, En vivo Metodología

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Una aplicación Novel of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Protocolo de ensayo basado en células para la predicción pronóstica de la escoliosis idiopática del Uso de Celular Espectroscopía Dieléctrica

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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