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 mirror and 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 re-localization 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 mirroring 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. 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 4 to 5 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. 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. 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 windows 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 5-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 a cerulein treatment.

Labial 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.