Name: Cirugía de ventana pancreática murina
Uploaded: 2023-10-06T13:20:00
Description: Watch this Scientific Journal Video about Murine Pancreatic Window Surgery at JoVE.com

murine pancreatic window surgery

Imágenes pancreáticas murinas con ventana estabilizada implantada

Benign and malignant pancreatic diseases are potentially life-threatening, with considerable gaps in the understanding of their pathophysiology. Pancreatitis-inflammation of the pancreas-is the third major cause of gastrointestinal disease-related hospital admissions and readmissions in the US and is associated with substantial morbidity, mortality, and socioeconomic burden1. Ranked as the third leading cause of cancer-related death2, pancreatic ductal adenocarcinoma (PDAC) accounts for most pancreatic malignancies3 and portends a poor 5-year survival rate of only 11%2. The leading cause of cancer-related mortality in PDAC is overwhelming metastatic burden. Unfortunately, most patients present with metastatic disease. Therefore, understanding the dynamics of metastasis in PDAC is a critical unmet need in the field of cancer research.
The mechanisms underpinning inflammation and the metastatic cascade of the pancreas are poorly understood. A major contributor to this gap in knowledge is the inability to observe pancreatic cellular dynamics in vivo. Direct observation of these cellular dynamics promises to unveil critical targets to leverage and improve the diagnosis and treatment of those with pancreatic disease.
Intravital imaging (IVI) is a microscopy technique that allows researchers to visualize and study biological processes in living animals in real time. IVI allows high-resolution, direct visualization of intracellular and microenvironmental dynamics in vivo and within the native environment of the biological process in question. Therefore, IVI allows in vivo observation of healthy and pathologic processes.
Contemporary whole-body imaging modalities such as MRI, PET, and CT offer excellent views of entire organs and can reveal pathologies, even before the onset of clinical symptoms4. They are unable, however, to attain single-cell resolution or reveal the earliest stages of disease-pancreatitis or malignancy.
Previous research has used single-cell resolution IVI to observe benign and malignant diseases of skin5,6, breast7, lung8, liver9, brain10, and pancreatic tumors11, leading to insights into mechanisms of disease progression12. However, the murine pancreas poses significant obstacles to achieving single-cell resolution using IVI, primarily due to its deep visceral location and high compliance. Moreover, it is a branched, diffusely distributed organ within the mesentery that connects to the spleen, small intestine, and stomach, making it challenging to access. The tissue is also highly sensitive to motion caused by adjacent peristalsis and respiration. Minimizing movement of the pancreas is essential for single-cell resolution microscopy, as motion artifacts of even a few microns can blur and distort images, making tracking the dynamics of individual cells impossible13.
To perform IVI, an abdominal imaging window (AIW) must be surgically implanted9,11. To implant the AIW surgically, a metal window frame is sutured into the abdominal wall. Afterward, the organ of interest is attached to the frame using cyanoacrylate adhesive. While this is sufficient for some rigid internal organs (e.g., liver, spleen, rigid tumors), attempts at imaging the healthy murine pancreas are compromised by suboptimal lateral and axial stability due to the tissue&#39;s compliant texture and complex architecture14. To address this limitation, Park et al.14 developed an imaging window specifically designed for the healthy pancreas. This Pancreas Imaging Window (PIW) minimizes the influence of intestinal movement and breathing by incorporating a horizontal metal shelf within the window frame, just below the coverslip, stabilizing the tissue and maintaining its contact with the cover glass. While the PIW offers increased lateral stability, we found that this window still demonstrates axial drift and additionally prevents the imaging of large solid tumors due to the narrow gap between the metal shelf and coverslip15.
To address these limitations, we developed the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP), an implantable imaging window capable of achieving stable long-term imaging of both the healthy and diseased pancreas (Figure 1)15. Here, we provide a comprehensive protocol for the surgical procedure used to implant the SWIP. Although the primary objective was to study the dynamic mechanisms involved in metastasis, this method can also be utilized to explore various aspects of pancreas biology and pathology.

Autor destacado: Revolucionando la comprensión de la enfermedad pancreática a través de imágenes intravitales avanzadas 

Ventana estabilizada para la obtención de imágenes intravitales del páncreas murino

Employing high-resolution intravital imaging and an improved method of pancreas stabilization, we aim to enhance comprehension of the physiological dynamics of pancreatic adenocarcinoma, pancreatitis, and the healthy pancreas. This approach makes it possible to address gaps in the pathophysiological understanding, potentially advancing treatment strategies for these high-risk conditions. Single-cell resolution intravital imaging has illuminated the physiology and mechanisms of disease progression in various tissues.However, the pancreas presents unique challenges due to its deep placement, compliance, and susceptibility to motion artifacts, hindering effective access for observation for both benign and malignant conditions. Our SWIP technique enhances murine pancreas stabilization compared to prior abdominal imaging windows. The uniquely designed frame features etched lines for the use of micro-cartography, which enables consistent region relocalization across multiple imaging sessions.This facilitates the measurement of subcellular dynamics over days, providing valuable insights. The SWIP protocol enables stable, high-resolution, single-cell intravital imaging of the murine pancreas in normal and diseased states. It's especially beneficial for prolonged 3D and 4D imaging, offering insights into cellular interactions and disease mechanisms.Invaluable for unraveling pancreatic physiology and pathology, this technique visualizes single cells and their context in vivo.

Watch this Scientific Journal Video about Murine Pancreatic Window Surgery at JoVE.com

Cirugía de ventana pancreática murina

Comience colocando un ratón anestesiado sobre su lado derecho sobre un soporte quirúrgico calentado para exponer su abdomen izquierdo. Ancla la parte delantera y las extremidades traseras del ratón con cinta de papel, asegurando la visibilidad del bazo. A continuación, desinfecte la piel en el sitio quirúrgico aplicando generosamente un antiséptico.Confirme que el ratón está completamente anestesiado administrando un pellizco en el dedo del pie. Con ayuda de unas pinzas, levante la piel de la parte superior izquierda del abdomen y realice una incisión circular de 10 milímetros en la capa de piel y músculo con unas tijeras castroviejas. Localice el páncreas unido al bazo para determinar la dirección en la que se debe colocar el punto de cruz.Use una sutura de seda 5/0 para colocar la primera puntada en la capa muscular en el lugar identificado. Asegure el extremo con tres a cinco nudos. Continúe suturando directamente a través de la incisión.Luego corta y deja una cola de aproximadamente cinco centímetros. Repite la costura, esta vez perpendicular a la puntada original. A continuación, levante con cuidado y coloque el páncreas sobre el punto de cruz.Coloca una puntada de cuerda de bolso aproximadamente a un milímetro del agujero, entrelazando la piel y la capa muscular. Coloque el marco de la ventana de modo que los bordes de la incisión circular se encuentren dentro de la ranura de la ventana. Ahora, cierre la ventana implantada atando firmemente la seda 5/0.Cargue 100 microlitros de adhesivo de cianoacrilato líquido en una jeringa de 10 milímetros. Aplique un chorro delicado de aire comprimido hacia el pañuelo durante unos 10 segundos para secarlo. Con unas pinzas, sujete el marco de la ventana por su borde exterior y levántelo con cuidado para asegurar la separación del páncreas de la base del marco de la ventana.Dispensar una fina capa de adhesivo de cianoacrilato líquido a lo largo del hueco de la ventana, evitando el tejido del páncreas. A continuación, utilice pastillas de vacío para levantar el cubreobjetos de cinco milímetros. Coloque suavemente el cubreobjetos en el centro del marco de la ventana óptica y mantenga una ligera presión durante aproximadamente 25 segundos para permitir que el adhesivo se asiente.Con unas pinzas, separe el cubreobjetos de las pastillas de vacío. A continuación, apriete las suturas de punto de cruz para asegurar el páncreas al cubreobjetos y corte los extremos de la sutura. Finalmente, retire la cinta del mouse.Apague el vaporizador de isoflurano y vuelva a colocar el ratón en una jaula limpia. Se observó un aumento de la estabilidad lateral y axial del tejido pancreático después del período de asentamiento. Las imágenes con el SWIP mostraron niveles más bajos de deriva en comparación con las ventanas de imágenes abdominales y de páncreas.Se realizaron imágenes seriadas del páncreas murino después del tratamiento con ceruleína. La reubicación de los lóbulos se observó después de dos días consecutivos. El SWIP también se puede utilizar para la obtención de imágenes a largo plazo, lo que permite la visualización y cuantificación de la motilidad de la vacuola.Por ejemplo, la velocidad promedio de las vacuolas en el páncreas murino aumentó en aproximadamente un 10% después del tratamiento con ceruleína, en relación con el PBS. El tratamiento con ceruleína también aumentó la frecuencia media de giro de las estructuras subcelulares en un 10%Sin embargo, no hubo diferencias significativas en la velocidad neta, la direccionalidad o la distancia acumulada entre los tratamientos. SWIP permite la visualización y captura de la migración de células tumorales.Se observó la migración colectiva de células tumorales en racimos durante períodos cortos de tiempo. También se observó la migración unicelular de células tumorales y macrófagos.

Research

JoVE Journal

Cancer Research

Murine Pancreatic Window Surgery

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...