Name: Impianto di tumore al pancreas nei topi
Uploaded: 2023-10-06T13:20:00
Description: Watch this Scientific Journal Video about Pancreas Tumor Implantation in Mice at JoVE.com

pancreas tumor implantation in mice

Imaging pancreatico murino utilizzando una finestra stabilizzata impiantata

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.

Autore in primo piano: Rivoluzionare la comprensione della malattia pancreatica attraverso l'imaging intravitale avanzato 

Finestra stabilizzata per l'imaging intravitale del pancreas 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 Pancreas Tumor Implantation in Mice at JoVE.com

Impianto di tumore al pancreas nei topi

Iniziare preparando aliquote di cellule tumorali KPC singeniche alla concentrazione richiesta. Conservare questa sospensione cellulare in una siringa da insulina su ghiaccio. Successivamente, trasferire un topo anestetizzato in un cappuccio chirurgico sterile e posizionarlo in una posizione di decubito laterale destro parziale.Fissare gli arti con del nastro di carta. Sterilizzare l'addome del topo con un antisettico. Usa le pinze e le forbici castroviejo per eseguire un'incisione della pelle sottocostale sinistra lunga da 10 a 15 millimetri.Controllare l'emostasi se necessario utilizzando tamponi di cotone. Usa una penna per cauterizzazione per cauterizzare i vasi. Quindi, dividi il muscolo sottostante per entrare nel peritoneo.Esternalizzare il pancreas e la milza con tamponi di cotone sterili per prevenire danni ai tessuti. Allargare il pancreas verso l'esterno per evitare che si pieghi. Identificare il punto di iniezione del tumore nel corpo o nella coda del pancreas.Dopo un attento posizionamento del pancreas, tenere il tessuto con una pinza e inserire la punta della siringa da insulina di circa quattro o cinque millimetri con il lato smussato rivolto verso l'alto. Quindi, iniettare lentamente la soluzione di cellule tumorali. Un'iniezione riuscita sarà evidente dalla formazione di una piccola bolla.Concludi la procedura riportando con cura il pancreas nell'addome, facendo attenzione a non disturbare la bolla. Dopo aver chiuso il sito dell'incisione, riportare il topo in una gabbia pulita posta sotto una lampada riscaldante. Somministrare antibiotici nell'acqua potabile per prevenire qualsiasi infezione.Lasciare che il tumore si sviluppi per un periodo di 10-14 giorni fino a quando non è palpabile attraverso la parete addominale.

Research

JoVE Journal

Cancer Research

Pancreas Tumor Implantation in Mice

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 ...