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 soft tissues, even beyond that of titanium16.
<ol>
	<li>Start the passivation process by washing the optical window frames with a 1% (w/v) enzymatically-active detergent solution.</li>
	<li>Submerge the frames in a 5% (w/v) sodium hydroxide solution at 70 &#176;C for 30 min inside a glass jar.</li>
	<li>Take out the frames and rinse them with deionized water.</li>
	<li>Immerse the frames in a 7% (w/v) citric acid solution at 55 &#176;C for 10 min inside a new glass jar.</li>
	<li>Remove the frames and rinse them with deionized water again.</li>
	<li>Repeat step 1.2, and finally, rinse the window frames with deionized water one last time.</li>
</ol>
2. Preparation for tumor implantation or window surgery
NOTE: For studies of pancreatic tumors, tumor cells must be implanted and allowed to grow into overt tumors. To visualize the tumor cells in vivo,&#160;it is recommended to use cells that have been genetically altered to express fluorescent proteins such as Dendra2. Using fluorescent protein labels that are bright will mitigate potential issues with tissue autofluorescence. Other potential fluorescent proteins, dyes, and genetically encoded fluorescent mouse models that may be used have been discussed elsewhere17,18. To prevent contamination of the operative field, perform the surgical procedure in a hood or laminar flow cabinet and ensure that distinct areas are used for preparation, surgery, and recovery.
<ol>
	<li>Prior to the surgery, sterilize all surgical instruments in an autoclave and, if necessary, use a hot bead sterilizer for subsequent procedures. Ensure that the surgery employs a tips-only technique.</li>
	<li>Switch on the heated surgical pad and bead sterilizer and wait for it to reach the appropriate operating temperature. The heating pad temperature should be monitored with a surface thermometer to avoid potential burns. Place a sterile cloth over the heating pad if the temperature cannot be adequately controlled. 
	NOTE: Body temperature during short procedures (&#8804;20 min), such as tumor and window implantation, is minimally affected while using a heated surgical pad. However, longer periods of anesthesia, such as during extended timelapse imaging, require the mouse to be placed in a heated chamber to maintain body temperature.</li>
	<li>Anesthetize the mouse with 5% isoflurane in an anesthesia chamber.</li>
	<li>Critical Step: Lower the anesthesia to 2% once the mouse is unconscious. Carefully monitor the anesthesia level and the mouse&#39;s vitals (e.g., using a pulse oximeter)19.</li>
	<li>Place a small drop of eye lubricant on each eye of the mouse to prevent corneal drying.</li>
	<li>Prior to surgery, apply depilatory cream generously to the left upper abdomen to remove hair. After 20 s, use moistened tissue paper to firmly wipe away the hair and depilatory cream. Repeat the process as needed until all hair is removed from the surgical area.</li>
	<li>Inject 10 &#181;L of buprenorphine (0.1 mg/kg) diluted in 90 &#181;L of PBS subcutaneously to ensure preoperative analgesia.</li>
</ol>
3. Pancreas tumor implantation
<ol>
	<li>Prepare aliquots of tumor cells at the desired concentration (based on tumor cell doubling time). Place the cell suspension in an insulin syringe and keep it on ice. To follow this protocol, use 106 syngeneic KPC tumor cells20 suspended in a maximum of 50 &#181;L of PBS, following the orthotopic injection protocol adapted from Erstad et al.21 
	NOTE: This cell line injected at this concentration routinely produced palpable or appropriately large tumors by 10-14 days. Subclones of this cell line and other pancreatic cell lines would need to be evaluated for appropriate concentrations and timelines to produce appropriately sized tumors).</li>
	<li>Wash hands using antiseptic soap.</li>
	<li>Prior to each new surgery, put on new sterile gloves.</li>
	<li>Transfer the mouse to the sterile surgical hood and place it in a partial right lateral decubitus position.</li>
	<li>Secure the limbs with paper tape. 
	NOTE: Proper use of the instruments is important throughout the procedure. Examples of how to hold forceps, Castroviejo scissors, and the vacuum pickup tool are shown in Figure 2A-C.</li>
	<li>Sterilize the abdomen with antiseptic (Figure 2D).</li>
	<li>Ensure the animal is fully anesthetized by performing a toe pinch test.</li>
	<li>Make a 10-15 mm left subcostal incision in the skin using forceps and Castroviejo scissors (Figure 2E).</li>
	<li>Control hemostasis using cotton swabs or a cautery pen when/where deemed necessary.</li>
	<li>Carefully divide the underlying muscle with forceps and Castroviejo scissors to enter the peritoneum (Figure 2F).</li>
	<li>Using sterile cotton swabs, atraumatically externalize the pancreas and spleen.</li>
	<li>Splay the pancreas out so there are no folds (Figure 2G).</li>
	<li>Identify the desired tumor injection site in the body or tail of the pancreas (away from blood vessels).</li>
	<li>Critical step: After careful positioning of the pancreas, use forceps to provide tension to the tissue and insert the insulin syringe tip, with the bevel facing upwards, into the desired site of the pancreas to a depth of 4-5 mm (Figure 2H).</li>
	<li>Slowly inject the tumor cell solution. Look for a small bubble that confirms a successful injection (Figure 2I).</li>
	<li>Carefully return the pancreas to the abdomen without disturbing the tumor cell injection bubble (Figure 2J).</li>
	<li>Using absorbable 5-0 polydioxanone sutures, close the muscle layer first and&#160;then the skin with&#160;interrupted sutures&#160;(Figure 2K-N).</li>
	<li>Cover the incision with cyanoacrylate glue (Figure 2O), then return the mouse to a clean cage under a heating lamp for recovery. Administer antibiotics in drinking water to prevent infection. Monitor the mice and allow them to completely recover from surgery. 
	NOTE: Antibiotics are administered as required by IACUC protocol. All animals are housed individually.</li>
	<li>Allow the tumor to develop for 10-14 days until it is palpable through the abdominal wall.</li>
</ol>
4. Pancreas window surgery
<ol>
	<li>When the animals are ready for imaging, begin the window implantation surgery. To begin, wash hands with antiseptic soap.</li>
	<li>Before every new surgery, put on fresh sterile gloves.</li>
	<li>On the heated surgical stand, place the mouse in the right lateral decubitus position to expose the left abdomen.</li>
	<li>Anchor the mouse&#39;s front and hind limbs to the heated surgical stage cranially and caudally using paper tape. Ensure the spleen (beneath the skin) is visible within the surgical field (Figure 3A).</li>
	<li>To maintain sterility, unpackage all surgical instruments in the hood.</li>
	<li>Disinfect the surgical site by swabbing the mouse&#39;s skin with a generous application of antiseptic.</li>
	<li>Ensure the animal is fully anesthetized by performing a toe pinch test.</li>
	<li>Critical step: Lift the skin of the left upper quadrant of the abdomen with forceps and make a ~10 mm circular incision in the skin and musculature using Castroviejo scissors (Figure 3B,C).</li>
	<li>Control the bleeding and maintain hemostasis using cotton swabs or the cautery pen, where needed.</li>
	<li>Localize the pancreas, which is attached to the spleen, and identify the direction the pancreas is laying within the incision to decide where the supporting cross-stitch should be placed.</li>
	<li>Using 5-0 silk suture, place the first stitch at the desired location in the muscle layer. Tie this end with 3-5 knots. (Figure 3D,E)</li>
	<li>Continue to stitch directly across the incision. Cut and leave a tail of ~5 cm (Figure 3F).</li>
	<li>Repeat steps 4.11 and 4.12 perpendicular to the first stitch (Figure 3G,H).</li>
	<li>Critical step: Gently lift and position the pancreas over the cross-stitch (Figure 3I,J). Take care not to damage the pancreas during manipulation.</li>
	<li>Critical step: Using the 5-0 silk suture, perform a purse-string stitch ~1 mm from the hole, circumferentially, interlacing the skin and muscle layer (Figure 3K).</li>
	<li>Position the window frame so the edges of the circular incision are seated within the window&#39;s groove (Figure 3L).</li>
	<li>Fasten the implanted window by firmly tying down the 5-0 silk.</li>
	<li>Load 100 &#181;L of liquid cyanoacrylate adhesive into the 1 mL syringe.</li>
	<li>Dry the tissue by applying a delicate flow of compressed air for ~10 s.</li>
	<li>Clasp the window frame by its outer edge with forceps and gently raise it to ensure separation of the pancreas from the undersurface of the window frame.</li>
	<li>Critical step: Dispense a thin layer of liquid cyanoacrylate adhesive along the recess of the window (Figure 3M). Be sure not to get any of the adhesive on the pancreas tissue.</li>
	<li>Using vacuum pickups, lift the 5 mm coverslip.</li>
	<li>Carefully place the coverslip inside the recess in the center of the optical window frame. Hold with light pressure, allowing the cyanoacrylate adhesive to set (~25 s).</li>
	<li>Separate the coverslip from the vacuum pickups using forceps.</li>
	<li>Tighten the cross-stitch sutures to secure the pancreas snugly to the coverslip (Figure 3N,O). Note: Do not overtighten the cross-stitch as it can cause damage and ischemia to the pancreas.</li>
	<li>Cut the ends of the suture.</li>
	<li>Strip off the tape from the mouse.</li>
	<li>Switch off the isoflurane vaporizer.</li>
	<li>Relocate the mouse to a clean cage or directly to the intravital microscope.</li>
	<li>House the animals individually following window&#160;surgery and monitor them&#160;until full recovery.</li>
	<li>Imaging is then carried out on a two-laser multiphoton microscope as we have previously described 22,23,24 For long imaging sessions the mouse is placed in a heated chamber to maintain body temperature and provided with supporting fluids as per IACUC standards.</li>
</ol>
5. Cerulein treatment for the induction of pancreatitis
<ol>
	<li>To investigate the onset of pancreatitis, treat healthy mice with cerulein after implantation of the SWIP. Ensure that the mice are fasted for 14-18 h and given ad libitum water before cerulein administration.</li>
	<li>Inject 50 &#181;g/kg of cerulein in 100 &#181;L of sterile 1x DPBS intraperitoneally at 1 h intervals for up to eight injections. Administer an equivalent volume of 1x DPBS alone, injected intraperitoneally, to the control mice.</li>
	<li>Following imaging, sacrifice the mice 24 h after the first injection by cervical dislocation as per IACUC standards.</li>
	<li>Perform imaging on a two-laser multiphoton microscope as previously described22,23,24. For long imaging sessions, place the mouse in a heated chamber to maintain the body temperature and provide it with supporting fluids as per IACUC standards.</li>
</ol>

Murine Pancreatic Imaging Using Implanted Stabilized Window

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The authors have no conflicts of interest to disclose.

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><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&amp;nbsp;</td></tr><tr><td>7% (w/v) solution of citric acid</td><td>Sigma-Aldrich&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;5287-120V</td><td>Bead Sterilizer</td></tr><tr><td>Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td><td>Roboz Surgical</td><td>RS-5135&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Fisher Scientific</td><td>867WCNOGLUE</td><td>Cotton Swabs</td></tr><tr><td>SHARP Precision Barrier Tips, For P-100, 100 &amp;micro;L</td><td>Denville Scientific Inc.</td><td>P1125</td><td>100 &amp;micro;L Pipet Tips</td></tr><tr><td>Tetramethylrhodamine isothiocyanate&amp;ndash;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.

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

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

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.

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 ...

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

Research

JoVE Journal

Cancer Research

1.8K Views.  Einstein College of Medicine / Montefiore Medical Center. Door gebruik te maken van intravitale beeldvorming met hoge resolutie en een verbeterde methode voor pancreasstabilisatie, willen we het begrip van de fysiologische dynamiek van adenocarcinoom van de alvleesklier, pancreatitis en de gezonde alvleesklier verbeteren. Deze aanpak maakt het mogelijk om hiaten in het pathofysiologische begrip aan te pakken, waardoor mogelijk behandelingsstrategieën voor deze risicovolle aandoeningen worden bevorderd. Intravitale beeldvorming met eencellige resolu...

Video: Auteur in de schijnwerpers: een revolutie in het begrip van pancreasaandoeningen door middel van geavanceerde intravitale beeldvorming 

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Medicine

Pancreatic Intravital Imaging Window Installation in Mouse Model: An Integrated Pancreatic Imaging Window for Time-lapse In Vivo Imaging of Murine Pancreas Microstructure

Source: Park, I. et. al. <a href="https://www.jove.com/v/62538">Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to implant a pancreatic imaging window to separate the pancreas from the intestines for longitudinal time-lapse high-resolution imaging of the pancreas microstructure.

Source: Park, I. et. al. Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital ...

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

Intravital Imaging: A Method for Cellular Level Observation of the Mouse Pancreas Through a Stabilized Imaging Window

Begin by switching on the intravital microscope, including the laser power. To insert a vascular catheter, apply pressure on the proximal side of the tail with the index and third finger. If necessary, heat the tail with a lamp. Disinfect the tail vein with a 70% ethanol spray, then, insert a 30 gauge catheter into the lateral tail vein and visualize the regurgitation of blood in the PE-10 tube. Apply silk tape on the catheter to stabilize it.
Inject FITC dextran and TMR dextran, or other fluorescent probes, as appropriate, according to the combination of fluorescent probes. Insert a rectal probe to automatically control the body temperature with the homeothermic heating pad system. Then, insert the pancreatic imaging window prepared during intravital microscopy setup into the window holder. Transfer the mouse from the surgical platform to the imaging stage.
To perform intravital imaging, start with imaging the pancreas at a low magnification, such as four times, to scan the whole view of the pancreas in the pancreatic imaging window. Once the region of interest has been determined, switch to a higher magnification objective lens, like 20 times or 40 times, to perform cellular level imaging with lateral and axial resolution approximately 0.5 micrometers and 3 micrometers, respectively.
Perform z-stack or time-lapse imaging to observe the 3D structure or cellular level dynamics, such as cell migration.

Stabilized Window for Intravital Imaging of the Murine Pancreas

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