Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Este trabajo fue apoyado con fondos de la Midwestern University Colegio de Ciencias de la Salud Investigación Facilitación Grant, otorgado a EEH, y Midwestern University Oficina de Investigación y Programas Patrocinados Intramural Grant, otorgado a KJL. El apoyo adicional fue proporcionado por Affiliated Laboratories y Affiliated Dermatology. Agradecemos a Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel y Ali Zaidi por su asistencia técnica.

Toda la experimentación en muestras de tejidos humanos fue aprobada por Western IRB (WIRB Protocol # 20142461). El único criterio para la recolección de tejidos para estos procedimientos es un diagnóstico de SCC probado con biopsia. No se describieron criterios de exclusión en el protocolo IRB original, pero no se utilizaron muestras de pacientes con una enfermedad transmisible transmitida por la sangre conocida. El consentimiento informado se obtuvo antes de la recolección de tejido cutáneo, sangre y saliva. La ...

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A juicio del autor, este protocolo es el primero de su tipo que se centra en la adquisición clínica de muestras de tejido cutáneo en un enfoque rentable y rápido. Los pacientes sometidos a microcirugía de Mohs se programan típicamente durante bloques específicos del tiempo, y la colección se limita a estos períodos. La recolección de muestras involucra el esfuerzo del asistente médico involucrado en el cuidado del paciente, el histotecnólogo de Mohs que procesa las muestras tumorales y un miembro del persona...

La investigación sobre el cáncer y los biomarcadores se basa en un suministro de muestras de tejido humano de calidad, y la oferta limitada ha obstaculizado la investigación 1 , 2 . Muchos estudios dermatológicos están limitados por la oferta inadecuada, la calidad variable y los costos asociados con el uso de tejido humano. El costo de establecer un gran biobanco dedicado se ha estimado en aproximadamente dos millones de dólares 3 , y estos costos colocan el uso de tejido humano fuera del alcance de muchos investigadores. Además, el proceso de generación y almacenamiento de muestras de investigación plantea el riesgo de afectar las operaciones clínicas y retrasar la atención al paciente si no se ejecuta cuidadosamente. Se ha establecido un biorepository rentable, basado en clínicas, que se centra en las muestras de cáncer de piel siguiendo las mejores prácticas recomendadas y la validación de muestras 4 , 5 , 6 . <pEste protocolo se ha desarrollado en una clínica de dermatología que realiza un gran volumen de cirugías micrográficas de Mohs para eliminar el carcinoma de células escamosas (SCC), el carcinoma basocelular (CBC) y los cánceres de piel de melanoma. Los donantes voluntarios pueden ser reclutados de esta población de pacientes. Es importante establecer el biorepository en el sitio de la colección para capturar rápidamente el tejido y la sangre de pacientes consentidos sin retrasar el tratamiento. La recopilación de muestras de la misma clínica minimiza las variaciones en las técnicas de recolección y minimiza las variaciones en la calidad de las muestras, lo que puede ser problemático para las aplicaciones posteriores 7 , 8 . El objetivo de la técnica de cirugía micrográfica Mohs es asegurar que todo el tejido canceroso se elimina mientras se preserva el mayor número posible de tejidos sanos. El procedimiento implica la eliminación progresiva de capas delgadas de tejido tumoral. Cada capa sucesiva es suExaminado tologically (después de cryosectioning el tejido del tumor y de realizar la mancha de H &amp; E) por un dermatologist para determinar si todo el tejido del cáncer se ha quitado. La extirpación y examen de capas posteriores de tejidos se realiza mientras el paciente permanece en la oficina. Esta técnica se considera la mejor opción de tratamiento para SCC 9 . En este punto, la herida se cierra y, para mejorar la cicatrización y la apariencia estética, el tejido normal adyacente (ANT) es frecuentemente extirpado. Por lo tanto, este procedimiento quirúrgico para extirpar un tumor es idealmente adecuado para recoger tejidos histológicamente caracterizados para futuros estudios. El procedimiento de adquisición para obtener tejido tumoral, tejido adyacente normal, saliva y muestras de sangre ha sido diseñado para tener un impacto mínimo en las tareas normales del personal ( Figura 1 ). Asistentes médicos realizar la sangre dibuja mientras se prepara al paciente para el procedimiento. Después de completar el procedimiento de Mohs, el M<html> Ohs histotechnologist prepara más diapositivas histológicas de la muestra y transfiere el tejido al biorepository. Los costos asociados con el establecimiento del depósito biológico incluyen la compra de congeladores de crioconservación, la creación de un espacio de laboratorio clínico modesto y el desarrollo de un programa de seguimiento de inventario. <img alt="Figura 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figura 1: Secuencia de recolección de muestras y personal responsable. Tras el ingreso del paciente y el logro del consentimiento del paciente, el asistente médico recoge un hisopo bucal y realiza un drenaje de sangre. El dermatólogo y el asistente médico luego excisan el tumor y cierran la herida, durante el cual se recogen las muestras de SCC y ANT, respectivamente. Un técnico de laboratorio dedicado procesa la sangre y secciona las muestras de SCC y ANT para el cultivo de tejidos, la preservación y la entrada en el biorepository.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. La diversidad de muestras recogidas permite una variedad de enfoques experimentales ( Figura 2 ). Las muestras recogidas del paciente son hisopos bucales (la saliva también se puede recoger si es necesario), sangre entera y tejido extirpado. Los hisopos bucales y una muestra de la sangre entera se guardan, sin procesar, para el genotipado y el emparejamiento de tejidos. La sangre entera se separa en glóbulos blancos (WBC) y fracciones de plasma para análisis futuros. Después del procesamiento de Mohs, el tumor congelado se coloca directamente en nitrógeno líquido y se transfiere a un congelador a -80ºC. Las muestras frescas, viables de tejido tumoral y de ANT se cultivan usando modificaciones de las técnicas anteriores 10 , 11 y luego se crioconservan. Durante la recolección, el número de cada tipo de muestra se registra en una hoja de cálculo antes de<html> El programa de seguimiento de inventarios para facilitar el procesamiento preciso ( Tabla 1 ). <img alt="Figura 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figura 2: Esquema de la recolección y procesamiento de muestras de biorrepositores basados ​​en la clínica. Se recoge un hisopo bucal y una muestra de sangre del paciente y se almacena para genotipificación y emparejamiento de tejido aguas abajo. La sangre entera se procesa posteriormente para el aislamiento de glóbulos blancos (WBC) y el análisis de CTC, así como para la colección de plasma y el análisis de biopsia de líquido. El tejido excisado durante el procedimiento de Mohs se procesa histológicamente para fines de diagnóstico, después de lo cual las diapositivas histológicas pueden usarse experimentalmente para análisis inmunohistoquímicos adicionales. Siempre que la muestra de tejido extirpada sea lo suficientemente grande, una porción de tejido fresco se elimina y secciona para aislamiento de proteína y ARN y para el establecimiento de líneas celulares cultivadas.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Fecha de colección: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Paciente 1 </td><td> Paciente 2 </td><td> Paciente 3 </td><td> Paciente 4 </td><td> Paciente 5 </td></tr><tr><td> Iniciales y fecha de nacimiento </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Color de los ojos </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tipo de ejemplo </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Ubicación de la muestra </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Saliva </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WAgujero de sangre </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Plasma </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Viable </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tejido Normal Viable </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs Nitrógeno Líquido </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Nitrógeno líquido normal del tejido </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Diapositivas </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabla 1: Lista de verificación para registrar las colecciones de muestras. Los datos rastreados y registrados con cada muestra recogida incluyen iniciales del paciente, fecha de nacimiento y color de los ojos (para la tipificación de la piel), así como la locatiEn la extracción de la muestra. El número de muestras de saliva, los volúmenes de recolección de sangre y el número de especímenes de tejidos viables y conservados recolectados también se registran como referencias para asignaciones a usos posteriores. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Haga clic aquí para descargar este archivo.</a> Para validar los procedimientos de recolección de muestras, cada tipo de muestra se ha probado en aplicaciones descendentes. Utilizando modificaciones de las técnicas anteriores 12 , el tumor y la ANT se han utilizado con éxito en el aislamiento de proteínas y ARN y pueden usarse potencialmente para aislar el ADN. Los explantes viables establecidos a partir de las secciones de tejido se han evaluado mediante microscopía, mientras que las diapositivas histológicas almacenadas se han utilizado para inmunohistoquímica e inmunofluorescencia. Siguiendo el protocolo descrito aquí, es posible extender este modelo a otras clínicas de dermatología, otros tipos de tumores (como el melanoma), Y otras especialidades y prácticas quirúrgicas para proporcionar muestras de tejido humano para la investigación multifacética en cánceres humanos. Es probable que algunas modificaciones de este protocolo sean necesarias para otras prácticas pero, en principio, este protocolo es aplicable a cualquier práctica quirúrgica que descarte rutinariamente muestras de pacientes recolectadas durante el tratamiento del paciente.

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			<td>IX511F3</td>
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establishment of a clinic-based biorepository

La incidencia de cáncer de piel ( por ejemplo, carcinoma de células escamosas, carcinoma basocelular y melanoma) ha aumentado en los últimos años. Se espera que haya una demanda paralela de muestras tumorales cutáneas para estudios de investigación biomédica. Sin embargo, la disponibilidad de tejidos es limitada debido al costo de establecer un depósito biológico y la falta de protocolos disponibles para obtener muestras clínicas que no interfieran con las operaciones clínicas. Se estableció un protocolo para recolectar y procesar el tumor cutáneo y las muestras asociadas de sangre y saliva que tienen un impacto mínimo en los procedimientos clínicos de rutina en la fecha de la cirugía de Mohs. Las muestras tumorales se recogen y procesan a partir de pacientes sometidos a su primera capa de cirugía Mohs para neoplasias cutáneas probadas por biopsia por el histotecnólogo Mohs. El tejido normal adyacente se recoge en el momento del cierre quirúrgico. Las muestras adicionales que se pueden recoger son hisopos de sangre entera y bucal. Mediante la utilización de muestras de tejido que normalmente se desechan, se generó un depósito biológico que ofrece varias ventajas clave basándose en la clínica frente al laboratorio. Estos incluyen una amplia gama de muestras recogidas; Acceso a los registros de pacientes no identificados, incluidos los informes de patología; Y, para el donante típico, el acceso a muestras adicionales durante las visitas de seguimiento.

El tumor y la ANT se han utilizado con éxito en el aislamiento de proteínas y se han ensayado mediante transferencia Western. Como se muestra en la Figura 3 , se pueden detectar marcadores cutáneos de carcinoma de células escamosas (Muc1 14 , FHL- 15 y p40 16 , 17 ) en muestras de pacientes recogidas y almacenadas en nuestro depósito biológico clínico. <p...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

Los tumores cutáneos se descartan a menudo después de la cirugía micrográfica de Mohs. Se describe aquí un protocolo que permite al personal de soporte clínico procesar y almacenar eficazmente muestras de tumores cutáneos ( por ejemplo, carcinoma de células escamosas, carcinoma basocelular y melanoma) para aplicaciones de laboratorio aguas abajo sin interferir con las operaciones clínicas.

Establecimiento de un Biorepository basado en la Clínica

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Mínimamente Invasiva Establecimiento de murino ortotópico xenoinjertos vejiga

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Investigación

Medicina

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Establecimiento de Human epitelial Enteroids y Colonoids de Whole Tejidos y Biopsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establecimiento y caracterización de IU y ITUAC en un modelo de ratón

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Establecimiento de un defecto segmentario Modelo femoral de tamaño crítico en ratones estabilizado por osteosíntesis Plate

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Establecimiento de un modelo de ojos secos severos mediante dacrioadenectomía completa en conejos

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Establecimiento y evaluación de un modelo de enfermedad por injerto de vena porcina

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Establecimiento de un modelo de rata simple y eficaz para la obtención de imágenes intraoperatorias de la glándula paratiroides

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Cirugía de fístula arteriovenosa modificada para establecer el modelo de ratón de sobrecarga de volumen del VD

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Autor destacado: Establecimiento y confirmación de un modelo de ratón con sobrecarga de volumen ventricular derecho postnatal  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.