The Evelyn Lipper Charitable Foundation, the Gruss-Lipper Biophotonics Center, the Integrated Imaging Program for Cancer Research, an NIH T-32 Fellowship (CA200561), and a Department of Defense Pancreatic Cancer Research Program (PCARP) grant PA210223P1.

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC).
1. Passivation of windows
NOTE: Passivation of stainless steel cleans the metal of contaminants and creates a thin oxide layer that greatly increases the metal&#39;s biocompatibility with sof...

Murine Pancreatic Imaging Using Implanted Stabilized Window

<ol>
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The SWIP protocol described here provides an improved method of pancreas tissue stabilization by utilizing a cross-stitch basket technique. Early abdominal imaging windows (AIWs) enabled intravital imaging (IVI) of internal organs of the abdomen but did not adequately limit the movement of soft tissues such as the pancreas. In response, Park et al. developed a pancreas imaging window (PIW) that incorporates a horizontal metal shelf and allows improved stabilization of the pancreas tissue while maintaining contact with th...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% (w/v) solution of enzyme-active detergent</td>
			<td>Alconox Inc</td>
			<td>NA</td>
			<td>Concentrated, anionic detergent with protease enzymes for manual and ultrasonic cleaning</td>
		</tr>
		<tr>
			<td>5% (w/v) solution of sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td>Passivation reagent</td>
		</tr>
		<tr>
			<td>5 mm cover glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72296-05</td>
			<td>Round Glass Coverslips&#160;</td>
		</tr>
		<tr>
			<td>7% (w/v) solution of citric acid</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>251275</td>
			<td>Passivation reagent</td>
		</tr>
		<tr>
			<td>28G 1 mL BD Insulin Syringe</td>
			<td>BD</td>
			<td>329410</td>
			<td>Syringe for cell injection</td>
		</tr>
		<tr>
			<td>Baytril 100 (enrofloxacin)</td>
			<td>Bayer (Santa Cruz Biotechnology)</td>
			<td>sc-362890Rx</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Bench Mount Heat Lamp</td>
			<td>McMaster-Carr</td>
			<td>3349K51</td>
			<td>Heat lamp</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Covetrus North America</td>
			<td>059122</td>
			<td>Buprenorphine Analgesia</td>
		</tr>
		<tr>
			<td>Castroviejo Curved Scissors</td>
			<td>World Precision Instruments</td>
			<td>WP2220</td>
			<td>Scissor for cutting tissue</td>
		</tr>
		<tr>
			<td>C57BL/6J Mouse</td>
			<td>Jackson Laboratory</td>
			<td>000664&#160;</td>
			<td>C57BL/6J Mouse</td>
		</tr>
		<tr>
			<td>Chlorhexidine solution</td>
			<td>Durvet</td>
			<td>7-45801-10258-3</td>
			<td>Chlorhexidine Disinfectant Solution</td>
		</tr>
		<tr>
			<td>Compressed air canister</td>
			<td>Falcon</td>
			<td>DPSJB-12</td>
			<td>Compressed air for drying tissue</td>
		</tr>
		<tr>
			<td>Cyano acrylate - Gel Superglue</td>
			<td>Staples</td>
			<td>234790-6</td>
			<td>Skin Glue</td>
		</tr>
		<tr>
			<td>Cyano acrylate - Liquid Superglue</td>
			<td>Staples</td>
			<td>LOC1647358</td>
			<td>Coverslip Glue</td>
		</tr>
		<tr>
			<td>DPBS 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>DPBS for cerulein/cell injections</td>
		</tr>
		<tr>
			<td>Gemini Cautery Kit</td>
			<td>Harvard Apparatus</td>
			<td>726067</td>
			<td>Cautery Pen</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>CellPoint Scientific</td>
			<td>GER&#160;5287-120V</td>
			<td>Bead Sterilizer</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&#34; Length</td>
			<td>Roboz Surgical</td>
			<td>RS-5135&#160;</td>
			<td>Graefe Micro Dissecting Forceps</td>
		</tr>
		<tr>
			<td>Imaging microscope</td>
			<td>NA</td>
			<td>NA</td>
			<td>See Entenberg et al. 2011 [27]</td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>NA</td>
			<td>NA</td>
			<td>See Entenberg et al. 2011 [27]</td>
		</tr>
		<tr>
			<td>Isoethesia (isoflurane)</td>
			<td>Henry Schein Animal Health</td>
			<td>50033</td>
			<td>Isoflurane Anesthesia</td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td>Fisher Scientific</td>
			<td>06-666-A&#160;</td>
			<td>Kim Wipes</td>
		</tr>
		<tr>
			<td>Laboratory tape</td>
			<td>Fisher Scientific</td>
			<td>159015R</td>
			<td>Laboratory Tape</td>
		</tr>
		<tr>
			<td>Mouse Dissecting Kit</td>
			<td>World Precision Instruments</td>
			<td>MOUSEKIT</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Mouse Paw Pulse Oximeter Sensor</td>
			<td>Kent Scientific Corpo</td>
			<td>MSTAT Sensor-MSE</td>
			<td>Pulse Oximeter</td>
		</tr>
		<tr>
			<td>Mouse Surgisuite</td>
			<td>Kent Scientific</td>
			<td>SURGI-M04</td>
			<td>Heated platform</td>
		</tr>
		<tr>
			<td>Nair Hair Removal Lotion</td>
			<td>Amazon</td>
			<td>B001RVMR7K</td>
			<td>Depilatory Lotion</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>TechAir</td>
			<td>OX TM</td>
			<td>Oxygen</td>
		</tr>
		<tr>
			<td>PERMA-HAND Black Braided Silk Sutures, ETHICON Size 5-0</td>
			<td>VWR</td>
			<td>95056-872</td>
			<td>Silk Suture</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 1x</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>PhysioSuite System</td>
			<td>Kent Scientific</td>
			<td>PhysioSuite</td>
			<td>Heated Platform Controller</td>
		</tr>
		<tr>
			<td>Puralube</td>
			<td>Henry Schein Animal Health</td>
			<td>008897</td>
			<td>Eye Lubricant</td>
		</tr>
		<tr>
			<td>Puritan Nonsterile Cotton-Tipped Swabs&#160;</td>
			<td>Fisher Scientific</td>
			<td>867WCNOGLUE</td>
			<td>Cotton Swabs</td>
		</tr>
		<tr>
			<td>SHARP Precision Barrier Tips, For P-100, 100 &#181;L</td>
			<td>Denville Scientific Inc.</td>
			<td>P1125</td>
			<td>100 &#181;L Pipet Tips</td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine isothiocyanate&#8211;Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>T1287-500MG</td>
			<td>Vascular Label</td>
		</tr>
		<tr>
			<td>Window-fixturing plate</td>
			<td>NA</td>
			<td>NA</td>
			<td>Custom made plate for window placement on microscope stage. Plate is made of 0.008 in stainless steel shim stock. For dimensions of plate see Entenberg et al., 2018 [8].</td>
		</tr>
		<tr>
			<td>Window Frame</td>
			<td>NA</td>
			<td>NA</td>
			<td>The window is composed of a steel frame with a central aperture that accepts a 5 mm coverslip. A groove of 1.75 mm around the circumference of the frame provides space for the peritoneal muscle and skin layers to adhere to. See Entenberg et al., 2018 [8].</td>
		</tr>
	</tbody>
</table>

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 high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

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.

Figure 1,&#160;adapted from Du et al.15, shows image stills from a time-lapse IVI movie of the murine pancreas. Some tissue motion can be observed within the initial settling period (first hour of imaging, Figure 1A). However, with continued imaging after this settling period (&#62;75 min), we observed an increase in lateral and axial stability (Figure 1B). The comparison of the stability of the SWIP with the...

Watch this Scientific Journal Video about Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging at JoVE.com

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

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

stabilized-window-for-intravital-imaging-of-the-murine-pancreas

Research

JoVE Journal

Cancer Research

Pancreas Tumor Implantation in Mice

Begin by preparing aliquots of syngeneic KPC tumor cells at the required concentration. Store this cell suspension in an insulin syringe on ice. Next, transfer an anesthetized mouse to a sterile surgical hood and position it in a partial right lateral decubitus position.Secure the limbs with paper tape. Sterilize the mouse's abdomen with antiseptic. Use forceps and castroviejo scissors to make a 10 to 15-millimeter-long left subcostal skin incision.Control hemostasis as necessary by using cotton swabs. Use a cautery pen to cauterize the vessels. Next, divide the underlying muscle to enter the peritoneum.Externalize the pancreas and spleen with sterile cotton swabs to prevent tissue damage. Splay the pancreas out to prevent folding. Identify the tumor injection point in the body or tail of the pancreas.After careful positioning of the pancreas, hold the tissue with forceps and insert the insulin syringe tip about four to five millimeters with the bevel side up. Then, inject the tumor cell solution slowly. A successful injection will be evident by the formation of a small bubble.Conclude the procedure by carefully returning the pancreas to the abdomen, making sure not to disturb the bubble. After closing the incision site, return the mouse to a clean cage placed under a heating lamp. Administer antibiotics in drinking water to prevent any infection.Allow the tumor to develop for a period of 10 to 14 days until it is palpable through the abdominal wall.

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.

951 Views.  Einstein College of Medicine / Montefiore Medical Center.  We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

Murine Pancreatic Imaging Using Implanted Stabilized Window

Summary