Nuestra investigación es apoyada por el Programa de Investigación Intramural del NIH, el Instituto Nacional del Ojo.

Todos los equipos y los reactivos utilizados son estériles. El protocolo fue aprobado por el Cuidado de Animales y el empleo Comisión (ACUC) en el NEI / NIH (protocolo de estudio en animales NEI-553), y se realizó de acuerdo a las directrices del NIH y los reglamentos. 1. La producción de la angiogénesis que contiene el factor de pellets En esta parte, las pastillas que contienen el factor angiogénico de interés están preparados. <ol><li> Hacer el 10% (w / v) sucralfato con PBS. Almacenar a temperatura ambiente. </li><li> Hacer el 12% (w / v) de poli-HEMA con etanol absoluto. Almacenar a temperatura ambiente. </li><li> Hacer 1 mg / l solución de bFGF. Tienda de -80 ° C. </li><li> Para hacer 50 bolitas, mezclar 5 l de poli-HEMA, 1μl de sucralfato y 4 l de solución madre bFGF, vórtice a fondo. </li><li> Coloque un trozo de parafina limpia en una placa Petri, se exponen a la luz ultravioleta durante 15 minutos en una campana de cultivo de tejidos. Caída de 0,2 l de la mezcla de solución en la parafina de una pastilla. Por lo tanto, 10 l de la mezcla de solución debe producir 50 pastillas. Deje que las pastillas seco a temperatura ambiente durante 1-2 horas. Los gránulos secos se pueden almacenar en 4 ° C durante varios días, o -80 º C durante varios meses. </li><li> Al uso, palanca y recoger las bolitas con la punta de un par de pinzas. Tenga mucho cuidado de no romper o hacer que la primavera de distancia. </li></ol> 2. Realización de la córnea del ratón ensayo de bolsillo En esta parte, vamos a demostrar cómo hacer un bolsillo en la córnea del ratón, y cómo insertar una pastilla en el bolsillo. El factor angiogénico contenidas en el sedimento se dará a conocer poco a poco a los alrededores de la córnea (Fig. 1). En esta demostración, el uso de 8 semanas hembras C57/Bl6 ratones. <ol><li> El ratón está profundamente anestesiados de forma sistemática con la mezcla de ketamina (75 mg / kg) y xilazina (5 miligramos por kilogramo, la inyección ip). Esto evitará que el ratón en un estado de anestesia durante 30-60 min. Anestesia tópica (0,5% proparacaína HCl) se aplica a la córnea. Después de 5 minutos, fijar un ojo con un microscopio de disección. </li><li> Un corte muy suave se hace en el centro de la córnea 1,2 -1,4 mm del limbo corneal (como se ilustra en la Figura 1) con un cuchillo de von Graefe cataratas. Tenga mucho cuidado de no cortar a través de la córnea. </li><li> Un bolsillo se hace bajo la capa del epitelio de la córnea horizontal insertando el cuchillo en el centro de la córnea y la ampliación del cuchillo hacia el cuidado del limbo (Fig. 1). El tamaño de la bolsa tiene que ser lo suficientemente grande para dar cabida a uno de los factores angiogénicos que contienen pellets. Tenga mucho cuidado de no perforar a través de la córnea. </li><li> Introduzca una pastilla con un par de pinzas en el bolsillo poco a poco. Trate de hacer que la pastilla lo más plana posible. </li><li> Después de la implantación de la pastilla, un ungüento oftálmico antibiótico tópico se aplica en los ojos. Los instrumentos se limpian con algodón con alcohol al 70% entre los animales. </li><li> Mueve el ratón a un lugar caliente y seco y el monitor, hasta que es capaz de mantener una postura erguida y luego devolverlo a su jaula. Los analgésicos sistémicos (por ejemplo, la buprenorfina, 2 mg / kg, la inyección ip) y una pomada tópica con antibióticos (por ejemplo, gentamicina) se dan dos veces al día durante 2 días después de la cirugía y el ratón es de cerca de 3 días. Se espera que la curación completa dentro de las primeras 48 horas después de la cirugía. El ojo con el bolsillo se examina con un alcance con lámpara de hendidura biomicro entre los días 4-10 después de la implantación de pellets. </li></ol> 3. Los resultados representativos <ol><li> Siete días después de la implantación de pellets, el ratón se anestesia sistémica y tópica como se describe en 2.1. </li><li> Observar los vasos sanguíneos en la córnea inducidos por el factor angiogénico bajo un microscopio de disección. En la córnea tratados con vehículo, no el crecimiento de los vasos sanguíneos se puede observar (Fig. 2A, tratadas con vehículo córnea). En la córnea tratados con bFGF, la angiogénesis robusta se puede ver (Fig. 2B). El crecimiento de vasos sanguíneos pueden ser evaluados utilizando la longitud máxima de los vasos sanguíneos (de los vasos normales del limbo a la parte superior de los nuevos vasos), y las áreas de vaso sanguíneo total (Fig. 2B, delineado con una línea discontinua de color rojo en el bFGF tratados con la córnea). Otros métodos de cuantificación también se han descrito 2,5. </li></ol><img src="/files/ftp_upload/3077/3077fig1.jpg" alt="Figura 1" /> Figura 1. Una córnea. Normales con tinción H &amp; E. No existe en los vasos sanguíneos de una córnea normal. B. Hacer un bolsillo debajo de la capa del epitelio de la córnea. C. Insertar una bolita en el bolsillo. D. El factor angiogénico se libera de la pastilla, y forman nuevos vasos sanguíneos de los vasos límbicos normal. <img src="/files/ftp_upload/3077/3077fig2.jpg" alt="Figura 2" /> Figura 2. Resultado Representante cinco días después de implantación de la pastilla. A. Córnea con una pastilla que contiene el único vehículo. No hay sangre nueva formación de vasos en la córnea. Sólo los vasos preexistentes limbal normales son visibles. B. Córnea con una pastilla que contiene bFGF. Robusta nuevos vasos sanguíneos (punto-alineados) creció desde el pre-existentes vasos límbicos normal.

<ol>
	<li>Cao, Y., Linden, P., Shima, D., Browne, F., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+angiogenic+activity+and+hypoxia+induction+of+heterodimers+of+placenta+growth+factor/vascular+endothelial+growth+factor.">In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.</a> The Journal of clinical investigation. 98, 2507-2511 (1996).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nature. 2, 2545-2550 (2007).</li><li>Poche, R. A., Saik, J. E., West, J. L., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+as+a+transplantation+site+for+live+imaging+of+engineered+tissue+constructs.">The mouse cornea as a transplantation site for live imaging of engineered tissue constructs.</a> Cold Spring Harbor protocols. , 5416-54 (2010).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+is+dispensable+for+blood+vessel+growth+but+critical+for+their+survival,+and+VEGF-B+targeting+inhibits+pathological+angiogenesis.">VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. , 106-6152 (2009).</li><li>Tong, S., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+response+of+angiogenesis+to+basic+fibroblast+growth+factor+in+rat+corneal+pocket+assay:+I.+Experimental+characterizations.">Dose response of angiogenesis to basic fibroblast growth factor in rat corneal pocket assay: I. Experimental characterizations.</a> Microvascular research. 75, 10-15 (2008).</li><li>Muthukkaruppan, V., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+mouse+cornea.">Angiogenesis in the mouse cornea.</a> Science. 205, 1416-1418 (1979).</li><li>Ziche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+assay+for+angiogenesis.">Corneal assay for angiogenesis.</a> Methods in molecular medicine. 46, 131-142 (2001).</li><li>Ziche, M., Morbidelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+pocket+assay.">The corneal pocket assay.</a> Methods in molecular biology. 467, 319-329 (2009).</li></ol>

No hay conflictos de interés declarado.

La córnea del ratón de bolsillo modelo de ensayo fue descrito por primera vez en 1979 por Muthukkaruppan VR y Auerbach R 6. Protocolos detallados han sido publicados por diferentes laboratorios 2,3,7,8. Nuestro laboratorio ha modificado y utilizado este método en nuestros estudios durante muchos años 4. La córnea del ratón ensayo de bolsillo es un modelo relativamente simple, con una gran reproducibilidad. Otra ventaja de este modelo es que ya que la córnea normalmente no tiene va...

<table border="1"><tbody><tr><th> Equipo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Microscopio de disección </td><td> Mundial de Instrumentos de Precisión </td><td> PZMIV-BS </td></tr><tr><td> Von Graefe cuchillo de cataratas (1.5x25mm hoja, plano) </td><td> Ambler quirúrgica </td><td> 3401E </td></tr><tr><td> Dumont # 5 fórceps </td><td> Herramientas de Bellas Ciencia </td><td> 11252-50 </td></tr><tr><td> Con lámpara de hendidura </td><td> Zeiss </td><td> 30 SL-M </td></tr></tbody></table> Tabla 1. Equipos <table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Catálogo # </th><th> Comentarios </th></tr><tr><td> bFGF (factor de crecimiento de fibroblastos) </td><td> Peprotech INC </td><td> 100-18B </td><td></td></tr><tr><td> Sucralfato (sacarosa octasulfate-complejo de aluminio) </td><td> Sigma </td><td> S0652 </td><td> 10% en PBS </td></tr><tr><td> poli-HEMA (poli (2-hidroxietil metacrilato)) </td><td> Sigma </td><td> P3932 </td><td> 12% de etanol </td></tr></tbody></table> Tabla 2. Reactivos y materiales

a mouse model of the cornea pocket assay for angiogenesis study

Una córnea normal es clara de los tejidos vasculares. Sin embargo, los vasos sanguíneos pueden ser inducidas a crecer y sobrevivir en la córnea cuando potentes factores angiogénicos son administrados 1. Esta singularidad ha hecho el ensayo de bolsillo córnea uno de los modelos más utilizados para los estudios de la angiogénesis. La córnea compone de múltiples capas de células. Por tanto, es posible incluir una pastilla que contiene el factor angiogénico de interés en la córnea para investigar su efecto angiogénico 2,3. Aquí, le ofrecemos una demostración paso a paso de cómo (I) producir el factor angiogénico que contienen gránulos (II) incorporar la pastilla en la córnea (III) analizar la angiogénesis inducida por el factor angiogénico de interés. Ya que el factor de crecimiento de fibroblastos básico (bFGF) es conocido como uno de los factores angiogénicos más potentes 4, aquí se utiliza para inducir la angiogénesis en la córnea.

Watch this Scientific Journal Video about A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study at JoVE.com

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

La córnea es la única que carece de los tejidos vasculares. Sin embargo, el fuerte crecimiento de los vasos sanguíneos y la supervivencia puede ser inducida en la córnea por potentes factores angiogénicos. Por lo tanto, la córnea puede dar con nosotros en una herramienta valiosa para los estudios de angiogénesis. Este protocolo muestra cómo realizar el modelo de ratón de ensayo de córnea bolsillo y la forma de evaluar la angiogénesis inducida por factores angiogénicos utilizando este modelo.

Un modelo de ratón de la Prueba de bolsillo para el Estudio de la angiogénesis de córnea

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

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Research

JoVE Journal

Medicine

15.9K vistas.  National Eye Institute. La córnea es la única que carece de los tejidos vasculares. Sin embargo, el fuerte crecimiento de los vasos sanguíneos y la supervivencia puede ser inducida en la córnea por potentes factores angiogénicos. Por lo tanto, la córnea puede dar con nosotros en una herramienta valiosa para los estudios de angiogénesis. Este protocolo muestra cómo realizar el modelo de ratón de ensayo de córnea bolsillo y la forma de evaluar la angiogénesis inducida por factores angio...

Video: A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

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 ...

Investigación

Medicina

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Un modelo murino de la implantación de un stent en la arteria carótida para el Estudio de la reestenosis

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Desprendimiento de Retina modelo en roedores por inyección subretiniana de Hialuronato de Sodio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

El implante de gel de fibrina en pulmón de ratón para el Estudio de la angiogénesis pulmón específico

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Establecimiento de un modelo de xenoinjerto Humano mieloma múltiple en el pollo para estudiar el crecimiento del tumor, la invasión y la angiogénesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Un modelo de ratón de cirugía ortopédica para el estudio de la disfunción cognitiva postoperatoria y regeneración de los tejidos

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Cicatrización de heridas de abrasión epitelial corneal con rebabas Ocular como un modelo de córnea

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Evaluación de la angiogénesis terapéutica en un modelo murino de isquemia de las extremidades posteriores

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...