murine pancreatic window surgery

Murine Pancreatic Imaging Using Implanted Stabilized Window

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.

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

Stabilized Window for Intravital Imaging of the Murine Pancreas

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

Murine Pancreatic Window Surgery

Begin by positioning an anesthetized mouse on its right side on a heated surgical stand to expose its left abdomen. Anchor the mouse's front end and hind limbs with paper tape, ensuring spleen visibility. Next, disinfect the skin at the surgical site by applying antiseptic generously.Confirm that the mouse is fully anesthetized by administering a toe pinch. Using forceps, lift the skin of the upper left abdomen and make a 10-millimeter circular incision in the skin and muscle layer using castroviejo scissors. Locate the pancreas attached to the spleen to determine the direction in which the cross stitch should be placed.Use a 5/0 silk suture to place the first stitch in the muscle layer at the identified location. Secure the end with three to five knots. Continue to stitch directly across the incision.Then cut and leave a tail of approximately five centimeters. Repeat the stitching, this time perpendicular to the original stitch. Next, carefully lift and position the pancreas over the cross stitch.Place a purse string stitch approximately one millimeter from the hole, interlacing the skin and muscle layer. Position the window frame so that the circular incision edges are located within the window's groove. Now, fasten the implanted window by tightly tying down the 5/0 silk.Load 100 microliters of liquid cyanoacrylate adhesive into a 10-millimeter syringe. Apply a delicate stream of compressed air toward the tissue for about 10 seconds to dry it. Using forceps, clasp the window frame by its outer edge and lift carefully to ensure separation of the pancreas from the base of the window frame.Dispense a thin layer of liquid cyanoacrylate adhesive along the recess of the window, avoiding the pancreas tissue. Then use vacuum pickups to lift the five-millimeter cover slip. Gently position the cover slip in the center of the optical window frame and maintain light pressure for approximately 25 seconds to allow the adhesive to set.Using forceps, detach the cover slip from the vacuum pickups. Next, tighten the cross stitch sutures to secure the pancreas to the cover slip and cut the suture ends. Finally, remove the tape from the mouse.Switch off the isoflurane vaporizer and relocate the mouse to a clean cage. Increased lateral and axial stability of the pancreatic tissue was observed after the settling period. Imaging with the SWIP showed lower levels of drift compared to the abdominal and pancreas imaging windows.Serial imaging of the murine pancreas was performed after cerulein treatment. Lobule relocation was observed after two consecutive days. The SWIP can also be used for long-term imaging, allowing visualization and quantification of vacuole motility.For example, the average speed of vacuoles in the murine pancreas increased by approximately 10%after cerulein treatment, relative to PBS. Cerulein treatment also increased the average turning frequency of subcellular structures by 10%However, there was no significant difference in net speed, directionality, or cumulative distance between treatments. SWIP enables visualization and capture of tumor cell migration.Collective cluster cell migration of tumor cells was observed over short time periods. Single cell migration of tumor cells and macrophages was also observed.

Research

JoVE Journal

Cancer Research

288 visualizações.  Einstein College of Medicine / Montefiore Medical Center. Comece posicionando um camundongo anestesiado em seu lado direito em um suporte cirúrgico aquecido para expor seu abdome esquerdo. Fixe os membros dianteiros e posteriores do mouse com fita adesiva de papel, garantindo a visibilidade do baço. Em seguida, desinfete a pele no local da cirurgia aplicando antisséptico generosamente.Confirme se o rato está totalmente anestesiado administrando uma pinça do dedo do pé. Usando pinças, levante a pele do abdome superior esquerdo e faça u...