Name: stabilized longitudinal in vivo cellular-level visualization of the pancreas in a murine model with a pancreatic intravital imaging window
Uploaded: 2021-05-06T14:30:00.000+00:00
Duration: 6 min 52 s
Description: Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

This study was supported by grant No. 14-2020-002 from the SNUBH Research Fund and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1F1A1058381, NRF-2020R1A2C3005694).

All procedures described in this paper were conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals (2011)26 and approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology (KAIST) and Seoul National University Bundang Hospital (SNUBH).
1. Preparation of the window and other materials
<ol>
	<li>Custom design the pancreatic intravital imaging window to seclude the pancreas from the bowel in the abdominal cavity18 (Figure 1A,B). A detailed blueprint of the window is described in a supplementary figure of a previous study18.</li>
	<li>Use C57BL/6N mice, 8-12-week-old males, for intravital pancreatic imaging. Inject anti-CD31 antibody conjugated with an Alexa 647 fluorophore, 2 h prior to the imaging, for the purpose of vessel labeling18.</li>
	<li>For the islets study, prepare a transgenic mouse model in which the islets are tagged with a fluorescent reporter protein. Here, we utilized MIP-GFP, where green fluorescent protein was expressed under the control of the mouse insulin 1 gene promoter, which is active in the beta cells of all islets in the mouse24.</li>
	<li>For the pancreatic cancer study, prepare transgenic cancer cells tagged with a fluorescent reporter protein and BALB/C nude mice. In this study, the PANC-1 NucLight Red cells were used. PANC-1 cancer cells25 were labeled with the NucLight Red fluorescent probe.</li>
	<li>Sterilize all surgical tools, cover glass (with PEG or not), and imaging windows using an autoclave.</li>
	<li>Apply PEG coating to the cover glass to prevent inflammatory response and increase biocompatibility, which is suitable for long-term imaging.</li>
</ol>
2. Surgery
<ol>
	<li>Prepare a sterile surgical platform and sterilize the surfaces with 70% ethanol. 
	NOTE: For longitudinal imaging sessions, aseptic technique is essential.</li>
	<li>Anesthetize mice with a mixture of tiletamine/zolazepam (30 mg/kg) and xylazine (10 mg/kg). 
	NOTE: Use of tiletamine/zolazepam is recommended instead of ketamine because of its adverse effect of hyperglycemia. Optimal anesthesia should be selected for the purpose of the experiment9,27.</li>
	<li>Monitor the body temperature using a rectal probe with a homeothermic controlled heating pad.</li>
	<li>Shave the left flank of the mouse and apply three rounds of alternating alcohol and iodine-based scrub. 
	NOTE: If depilatory cream is used,&#160;do not leave longer than 1 minute to avoid chemical burn.</li>
	<li>Make a 1.5 cm incision on the left flank of the mouse and dissect the skin and muscle.</li>
	<li>Perform a purse-string suture with a black or nylon 4-0 suture in the incision margin.</li>
	<li>Use a micro retractor on the incision and gently expose the spleen.</li>
	<li>Carefully pool the spleen with ring forceps and identify the pancreas.</li>
	<li>Place the window at the flank of the mouse and pass the spleen and pancreas through the open space of the window.&#160;Manipulation of the pancreas has to be very gentile as it can cause bleeding or pancreatitis</li>
	<li>Gently place the pancreas on the plate of the imaging window; the spleen will be placed on the open space of the window.</li>
	<li>For the cancer cell study, inject PANC-1 NucLight Red (1.0 x 106 cells) directly into the pancreas. 
	NOTE: For imaging the orthotopic human pancreatic cancer xenografts, direct implantation of cancer cell such as PANC-1 or other human pancreatic cancer cell could be facilitated28. To visualize with intravital fluorescence microscopy, PANC-1 cells were transduced with red fluorescent protein using the NucLight Red lentiviral reagent that labels the nucleus29.&#160;Although maximal injection volume is uncertain, decrease the volume as possible to avoid the pressure effect in the pancreas.</li>
	<li>Apply drops of N-butyl cyanoacrylate glue on the margin of the imaging window.
	<ol>
		<li>To minimize the amount of the applied glue, use a 31 G catheter needle for the application. If the amount of the drop is large, then the tissue will unintentionally adhere to the window or cover glass.</li>
	</ol>
	</li>
	<li>Gently apply a 12 mm round cover glass to the margin of the imaging window.</li>
	<li>Pull the suture loop to fit into the lateral groove of the window and tie it three times.</li>
	<li>Cut the maximal proximal site of the knot&#160;to prevent the interruption of tight stitches when these mice are awake.</li>
	<li>Let mice recover from anesthesia (2-3 hours) and inject ketoprofen (5 mg/kg, subcutaneous in the loose skin at the base of neck or hip of the animal) for pain relief. Reassess for signs of pain every 12 hours and&#160;ketoprofen should be considered every 24 hours for 1-3 days. 
	&#8203;NOTE: Analgesia influences insulin secretion in response to glucose9. The choice and timing of analgesia must be individualized for the experimental purpose.</li>
	<li>Accomodate the mouse in the cage with sufficient bedding to avoid the contact of the window to the cage. Avoid housing the house with the mice without the window surgery.</li>
</ol>
3. Intravital imaging
<ol>
	<li>Turn on the intravital microscope including the laser power.</li>
	<li>Turn on the heating pad and set the homeothermic regulation to 37 &#176;C. 
	NOTE: Alternatively, use a passive heating pad or lamp with frequent control if there is no homeothermic regulation.</li>
	<li>Perform intramuscular anesthesia with a mixture of tiletamine/zolazepam (30 mg/kg) and xylazine (10 mg/kg). 
	NOTE: Use of tiletamine/zolazepam is recommended instead of ketamine because of its adverse effect of hyperglycemia. Optimal anesthesia should be selected for the purpose of the experiments9,27.</li>
	<li>Insert a vascular catheter for the injection.
	<ol>
		<li>Apply pressure on the proximal side of the tail with the index and third finger as an alternative to a tourniquet application. Heat the tail with a lamp if needed.</li>
		<li>Sterilize the tail vein with a 70% ethanol spray.</li>
		<li>Insert a 30 G catheter into the lateral tail vein. Regurgitation of blood will be visualized in the PE10 tube.</li>
		<li>Apply a silk tape on the catheter to stabilize it.</li>
		<li>Inject FITC/TMR dextran or other fluorescent probes (25 &#181;g of anti-CD31 conjugated with Alexa 647), as appropriate, according to the combination of fluorescent probes18. 
		NOTE: For fluorescent conjugated antibody probes, inject 2 h before the imaging session.</li>
	</ol>
	</li>
	<li>Transfer the mouse from the surgical platform to the imaging stage.</li>
	<li>Insert a rectal probe to automatically control the body temperature with the homeothermic heating pad system.</li>
	<li>Insert the pancreatic imaging window into the window holder prepared during the intravital microscopy setup (Figure 2). For an inverted microscope, a window holder might not be required.</li>
	<li>Perform intravital imaging.
	<ol>
		<li>For imaging the pancreas, start with a low magnification objective lens (e.g., 4x) for scanning the whole view of the pancreas in the pancreatic imaging window (recommended field of view: 2500 x 2500 &#181;m).</li>
		<li>After determination of the region of interest, switch to higher magnification objective lens (20x or 40x) to perform the cellular level imaging (recommended field of view: 500 x 500 &#181;m or 250 x 250 &#181;m). In this experiment, the lateral and axial resolution was approximately 0.5 &#181;m and 3 &#181;m, respectively.</li>
		<li>Perform z-stack or time-lapse imaging to observe the 3D structure or cellular-level dynamics, such as cell migration. 
		NOTE: For imaging the fluorescent protein expressing cells of transgenic animals (MIP-GFP), 30 s of intermittent 488 nm laser exposure with power up to 0.43 mW was tolerable without noticeable photobleaching or tissue damage. For imaging the fluorescent proteins labeled with Alexa 647, the 640 nm laser power up to 0.17 mW was tolerable without noticeable photobleaching or tissue damage. Prolonged excitation laser exposure with a power above this setting may lead to photobleaching or tissue damage by phototoxicity. Adjust the adequate gain and power to appropriately image the region of interest. Detailed setting of parameters in intravital microscopy must be individualized for each intravital microscopy prepared in the institute.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The protocol described here consists of intravital imaging of the pancreas using a novel supporting base integrated pancreatic intravital imaging window modified from an abdominal imaging window. Among the protocols described above, the first critical step is the implantation of the intravital pancreatic imaging window in the mouse. For the application of the glue in the window, it is important to apply the glue between the margin of the window and the cover glass, but not on the pancreatic tissue, as it may significantly interrupt intravital imaging. Not only the glue itself between the glass and tissue but also adjunct dust particles may induce light scattering during imaging if the glue is directly applied to the tissue. In addition, the application of the adhesive may have toxic and non-physiological effects on the pancreas.
The second step is the amount of pancreatic tissue placed on the metal-supporting base plate. Because the pancreatic tissue is a sheet-like structure, the volume of pancreatic tissue on the plate needs to be controlled. If too large a volume is placed on the plate, the glue applied to the margin of the ring might adhere to the tissue, and the mass effect might hamper the perfusion of the pancreas. On the other hand, if too small a volume is placed on there, the field of view that can be visualized might be limited. This protocol of surgery on the window may require several trials to meet a consistent standard.
For long-term imaging over a period of 3 weeks, the most concerning issue was potential damage to the pancreatic imaging window. Unintended destruction of the cover glass in the pancreatic imaging window could occur during the long-term observation period. To prevent this, the mouse with the window must be housed separately, and hard objects with sharp edges should be removed from the cage. The euthanasia of mice should be considered should the cover glass break, if there are the severe signs of inflammation near the window, or if the animal appears to be in distress. In our experience, mice with the pancreatic imaging window were able to eat and exercise normally when the recovery after the surgery was appropriate and no other complication was developed.
In our previous experience with the abdominal imaging window, we failed to acquire high-quality cellular level imaging as well as longitudinal tracking of the same spots over multiple days. Compared to the abdominal imaging window, which provides a diverse platform for various abdominal organs, the pancreatic imaging window is further specified for imaging the pancreas as well as other organs that are soft and easily influenced by movements such as mesentery, spleen, and small bowel. However, the liver and kidney might be unfeasible in the pancreatic imaging window because of the limited space.
While the combination of a fluorescent mouse, cells, and antibody probes enables the visualization of the dynamic interactions between endothelial cells and either the islets or cancer cells, the protocol described here could be reproduced with other compositions of fluorescent-labeled cells or molecular probes suitable for each respective condition. Furthermore, expansive applications integrated with our method are expected, such as the CpepSfGFP reporter mouse with insulin secretion9,30, AAV8-mediated gene delivery targeting reactive oxygen species (ROS)31, or orthotopic tumor model32,33,34 in which the tumor in situ can fully stimulate the tumor microenvironment, including tumorigenesis, development, and metastasis35. Furthermore, patient-derived xenograft models can also be studied using our platform36.
There are a few limitations to be addressed in this study. First, even when we utilized the metal base for stabilization, we were unable to determine the mechanical stress induced on the tissue by the base and cover glass, which could affect blood flow. However, as depicted in the above figures, intravenous injection of a fluorescence-conjugated antibody (CD31) or dextran adequately labeled the vessel with no distinguishable non-perfused area, suggesting a minimal impact of mechanical stress on the normal blood flow inside the pancreatic tissue. Second, adverse reactions due to the adhesive could not be assessed in the pancreatic tissue. Nevertheless, we attempted to avoid touching pancreas with adhesives as carefully as possible to avoid any additional effects. Third, as discussed above, the unintended impact of anesthetic agents might affect insulin sensitivity and secretion, as described in the previous study9,27. In our experience, a mixture of ketamine and xylazine induced hyperglycemia compared to the mixture of tiletamine, zolazepam, and xylazine. A further study investigating the effect of anesthesia on insulin secretion should be performed and proper anesthesia with minimal adverse effects should be selected according to each experiment. Fourth, imaging of the pancreas is focused on the tail portion, and imaging of the head portion of the pancreas could be limited with our window.
In summary, a stabilized longitudinal imaging of the pancreas at the cellular level for up to several weeks was facilitated by our imaging system integrated with the pancreatic intravital imaging window optimized for in vivo pancreas imaging. Because intravital imaging provides dynamic insights into cell biology, immunology, and tumor biology, this protocol could be a useful method for investigating the pathophysiology of various diseases involving the pancreas.

The pancreas is an abdominal organ with an exocrine function in the digestive tract and an endocrine function of secreting hormones into the bloodstream. High-resolution cellular imaging of the pancreas could reveal the pathophysiology of various diseases involving the pancreas, including pancreatitis, pancreatic cancer, and diabetes mellitus1. Conventional diagnostic imaging tools such as computed tomography, magnetic resolution imaging, and ultrasonography are widely available in the clinical field1,2. However, these imaging modalities are restricted to visualizing only structural or anatomical changes, while alterations at the cellular or molecular level cannot be determined. Given that molecular changes in diabetes mellitus or pancreatic cancer in humans can initiate more than 10 years prior to the diagnosis3,4, the detection of pancreatic diseases from their molecular transition during the latent period has the potential to provide an early diagnosis and a timely intervention. Thus, imaging that will overcome the limitations of resolution and provide valuable insights into the function will remarkably gain attention by providing early diagnosis of pancreatic cancer or advanced identification of the alteration of the islets during the progression of diabetes mellitus5.
In particular with the islets, nuclear imaging, bioluminescence imaging, and optical coherence tomography have been suggested as non-invasive islet imaging techniques6. However, the resolution of these methods is substantially low, with typical values ranging from several tens to hundreds of micrometers, offering a limited capability to detect changes at the cellular level in the islets. On the other hand, previous high-resolution studies of islets were performed under ex vivo7,8 (e.g., slicing or digestion of the pancreas), non-physiologic9 (e.g., exteriorization of the pancreas), and heterotopic conditions10,11,12 (e.g., implantation under the kidney capsule, inside the liver, and in the anterior chamber of the eye), which restricts their interpretation and clinical implications. If in vivo, physiologic, and orthotopic model of high-resolution imaging can be established, it will be a critical platform for the investigation of pancreatic islets.
Intravital imaging, which reveals the pathophysiology at a microscopic resolution level in a live animal, has recently received great attention13. Of the in vivo imaging methods, the development of an abdominal imaging window14, which implants a window into the abdomen of a mouse, has allowed the discovery of novel findings (i.e., a pre-micrometastasis stage of early liver metastasis15 and mechanism of stem cell maintenance in the intestinal epithelium16). Although the abdominal imaging window provides valuable results, the applications of this window for the pancreas and the resulting intravital imaging research based on diseases involving pancreas, have not been extensively investigated.
Unlike the well-defined solid organ characteristics of the human pancreas, the pancreas of a mouse is a diffusely distributed soft tissue-like structure17. Therefore, it is incessantly affected by physiological movements including peristalsis and respiration. A previous study on the application of an abdominal imaging window for the pancreas demonstrated that wandering occurred due to motion-artifacts induced by bowel movements18. Severe blurring was observed in the resulting averaged image, which impeded the visualization and identification of the microscale structures.
Herein, we describe the use of a novel supporting base integrated pancreatic intravital imaging window combined with intravital microscopy19,20 to investigate the longitudinal cellular level events in diseases involving the pancreas. In addition to a detailed description of the methodology in the previous study18, the extended application of pancreatic imaging window for various diseases involving the pancreas will be addressed in this paper. In this protocol, a custom-built video-rate laser-scanning confocal microscopy system was utilized as an intravital microscopy system. Four laser modules (wavelengths at 405, 488, 561, and 640 nm) were utilized as an excitation source, and four channels of emission signals were detected by photomultiplier tubes (PMT) through bandpass filters (BPF1: FF01-442/46; BPF2: FF02-525/50; BPF3: FF01-600/37; BPF4: FF01-685/40). Laser scanning consisted of a rotating polygonal mirror (X-axis) and a galvanometer scanning mirror (Y-axis) that enabled the video-rate scanning (30 frames per second). Detailed information about intravital microscopy has been described in the previous studies10,18,19,20,21,22,23.
In our previous islet study18, we successfully and stably imaged the islets in live mice using a transgenic mouse model (MIP-GFP)24 in which the islets were tagged with GFP. The method enabled high-resolution visualization of the changes in the islets over a period of 1 week. It also facilitated imaging of the same islets for up to 3 weeks, which suggests the feasibility of long-term studies of the pancreatic islets for the functional tracking or monitoring during the pathogenesis of diabetes mellitus18. Furthermore, we developed an orthotopic pancreatic cancer model in which fluorescent pancreatic cancer cells (PANC-1 NucLight Red)25 were directly implanted into the pancreas of the mouse. With the application of the pancreatic intravital imaging window, this model could be utilized as a platform for investigating the cellular and molecular pathophysiology in the tumor microenvironment of pancreatic cancer and for the therapeutic monitoring of novel drug candidates.

<table><tbody><tr><td>Alexa Fluor 647 Succinimidyl Esters (NHS esters)</td><td>Invitrogen</td><td>A20006</td><td>Fluorescent probe for conjugate with antibody</td></tr><tr><td>BALB/C Nude</td><td>OrientBio</td><td>BALB/C Nude</td><td>BALB/C Nude</td></tr><tr><td>BD Intramedic polyethylene tubing</td><td>BD Biosciences</td><td>427401</td><td>PE10 catheter for connection with needle</td></tr><tr><td>C57BL/6N</td><td>OrientBio</td><td>C57BL/6N</td><td>C57BL/6N</td></tr><tr><td>Cover glasses circular</td><td>Marienfeld</td><td>0111520</td><td>Cover glass for pancreatic imaging window</td></tr><tr><td>FITC Dextran 2MDa</td><td>Merck (Former Sigma Aldrich)</td><td>FD200S</td><td>For vessel identification</td></tr><tr><td>IMARIS 8.1</td><td>Bitplane</td><td>IMARIS</td><td>Image processing</td></tr><tr><td>Intravital Microscopy</td><td>IVIM tech</td><td>IVM-C</td><td>Intravital Microscopy</td></tr><tr><td>IRIS Scissor</td><td>JEUNGDO BIO &amp;amp; PLANT CO, LTD</td><td>S-1107-10</td><td>This product can be replaced with the product from other company</td></tr><tr><td>Loctite 401</td><td>Henkel</td><td>401</td><td>N-butyl cyanoacrylate glue</td></tr><tr><td>Micro Needle holder</td><td>JEUNGDO BIO &amp;amp; PLANT CO, LTD</td><td>H-1126-10</td><td>This product can be replaced with the product from other company</td></tr><tr><td>Micro rectractor</td><td>JEUNGDO BIO &amp;amp; PLANT CO, LTD</td><td>17004-03</td><td>This product can be replaced with the product from other company</td></tr><tr><td>Microforceps</td><td>JEUNGDO BIO &amp;amp; PLANT CO, LTD</td><td>F-1034</td><td>This product can be replaced with the product from other company</td></tr><tr><td>MIP-GFP</td><td>The Jackson Laboratory</td><td>006864</td><td>B6.Cg-Tg(Ins1-EGFP)1Hara/J</td></tr><tr><td>Nylon 4-0</td><td>AILEE</td><td>NB434</td><td>Non-Absorbable Suture</td></tr><tr><td>Omnican N 100 30G</td><td>B BRAUN</td><td>FT9172220S</td><td>For Vascular Catheter, Use only Needle part</td></tr><tr><td>PANC-1 NucLightRed</td><td>Custom-made</td><td>Custom-made</td><td>Made in laboratory</td></tr><tr><td>Pancreatic imaging window</td><td>Geumto Engineering</td><td>Custom order</td><td>Pancreatic imaging window - custom order</td></tr><tr><td>Physiosuite</td><td>Kent Scientific</td><td>PS-02</td><td>Homeothermic temperature controller</td></tr><tr><td>Purified NA/LE Rat Anti-Mouse CD31</td><td>BD Biosciences</td><td>553708</td><td>Antibody for in vivo vessel labeling</td></tr><tr><td>Ring Forceps</td><td>JEUNGDO BIO &amp;amp; PLANT CO, LTD</td><td>F-1090-3</td><td>This product can be replaced with the product from other company</td></tr><tr><td>Rompun</td><td>Bayer</td><td>Rompun</td><td>Anesthetic agent</td></tr><tr><td>TMR Dextran 65-85kDa</td><td>Merck (Former Sigma Aldrich)</td><td>T1162</td><td>For vessel identification</td></tr><tr><td>Window holder</td><td>Geumto Engineering</td><td>Custom order</td><td>Window holder - custom order</td></tr><tr><td>Zoletil</td><td>Virbac</td><td>Zoletil 100</td><td>Anesthetic agent</td></tr></tbody></table>

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.

Intravital microscopy combined with the supporting base integrated pancreatic intravital imaging window enables longitudinal cellular level imaging of the pancreas in a mouse. This protocol with the pancreatic intravital imaging window provides long-term tissue stability that enables the acquisition of high-resolution imaging to track individual islets for up to 3 weeks. As a result, mosaic imaging for an extended field of view, three-dimensional (3D) reconstruction of z-stack imaging, and longitudinal tracking of the same position can be achieved. In addition, our intravital microscopy provides four channels (405, 488, 561, and 647 nm) of acquisition, which enables simultaneous multiple cell visualization with their interactions.
For the preliminary imaging, the window was implanted in a C57BL/6N mouse with intravenously injected anti-CD31 antibody conjugated with an Alexa 647 fluorophore. Wide-area imaging (Figure 3A) and magnified 3D imaging (Figure 3B-D, Supplementary Video 1) of the pancreas were facilitated with this system. Pancreatic tissue was visualized with autofluorescence, and the adjacent vasculature labeled with the anti-CD31 antibody was identified. Oscillation due to either peristalsis or respiration was not identified, resulting in averaged imaging with a high signal-to-noise ratio (Figure 4). Acinar cells, which require visualization at a microscale resolution in the pancreas, were clearly visualized in the averaged images.
For imaging of the islets, a MIP-GFP mouse was utilized. Using the mosaic imaging method, a wide-field view with high-resolution imaging enabled the visualization of the islets with the adjacent vasculature (Figure 5). Approximately 40-50 islets were identified in the wide-field view. This stable imaging method could further facilitate the tracking of the islets for up to 3 weeks, as shown in a previous study (Figure 6)18.
For the cancer cell imaging, PANC-1 NucLight Red cells were directly implanted into the mouse pancreas during surgery (Figure 7). A dual-labeling strategy was used, consisting of PANC-1 NucLight Red cells and nearby vessels stained with anti-CD31 conjugated with Alexa 647. With our protocol, wide-field imaging of pancreatic cancer (Figure 7A), which delineates the margin of the tumor, and high-resolution 3D imaging at the single-cell level, was achieved (Figure 7B-D, Supplementary Video 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62538/62538fig01.jpg" /> 
Figure 1: Design and photograph of the pancreatic intravital imaging window. (A) A 3D and cross-sectional view of the pancreatic intravital imaging window. A detailed blueprint of the size and diameter is described in the previous paper18. (B) Anterior and posterior photograph of the pancreatic imaging window. Copyright 2020 Korean Diabetes Association from Diabetes Metab J. 2020 44:1:193-198. Reprinted with permission from The Korean Diabetes Association. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62538/62538fig02.jpg" /> 
Figure 2: Photograph of the implementation of the pancreatic intravital imaging window. A pancreatic intravital imaging window is implanted in the mouse in the XYZ translational stage and the imaging chamber holder attached to the tilting mount is connected to the pancreatic imaging window. Copyright 2020 Korean Diabetes Association from Diabetes Metab J. 2020 44:1:193-198. Reprinted with permission from The Korean Diabetes Association. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62538/62538fig03.jpg" /> 
Figure 3: Representative intravital pancreatic imaging in C57BL/6N mouse. (A) Wide-area image and (B) magnified 3D image of the pancreas (green) and its microvasculature (red) in the C57BL/6N mouse. Vessels are labeled with an anti-CD31 antibody conjugated with the Alexa 647 fluorophore. (C) 3D reconstructed image and (D) surface-rendering image of the mouse pancreas. Scale bar: 200 &#181;m (A) and 50 &#181;m (B-D). Also see Supplementary Video 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62538/62538fig04.jpg" /> 
Figure 4: Intravital imaging of acinar cell and adjacent vasculature. Acinar cells (green) and adjacent vasculature (red) in the C57BL/6N mouse. Vessels are labeled with Anti-CD31 antibody conjugated with the Alexa 647 fluorophore. Tissue stability accomplished with the pancreatic imaging window provides a high signal-to-noise ratio image. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62538/62538fig05.jpg" /> 
Figure 5: Representative intravital imaging of pancreatic islets in the MIP-GFP mouse. Wide-area mosaic and magnified image of the islets (green) and adjacent vasculature (red) processed with maximum intensity projection method in the pancreas of the MIP-GFP mouse. Vessels are labeled with an anti-CD31 antibody conjugated with the Alexa 647 fluorophore. Scale bar: 500 &#181;m (wide area) and 50 &#181;m (magnified). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62538/62538fig06.jpg" /> 
Figure 6: Longitudinal intravital imaging of islets in the pancreas of the MIP-GFP mouse. Longitudinal image of the islets for up to 3 weeks in the pancreas in the MIP-GFP mouse. Each arrowhead with different colors indicates the same islets. Scale bar: 100 &#181;m. Copyright 2020 Korean Diabetes Association from Diabetes Metab J. 2020 44:1:193-198. Reprinted with permission from The Korean Diabetes Association. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62538/62538fig07.jpg" /> 
Figure 7: Representative intravital imaging of the pancreatic cancer model. (A) Wide-area image and (B) magnified 3D image of the implanted PANC-1 NucLight Red cells (red) in the BALB/c Nude mouse. Vessels (blue) are labeled with an anti-CD31 antibody conjugated with the Alexa 647 fluorophore. (C) 3D reconstructed image and (D) surface-rendering image of pancreatic cancer in the mouse model. Scale bar: 500 &#181;m (A) and 50 &#181;m (B-D). Also see Supplementary Video 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/62538fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary video 1: In vivo 3D pancreatic imaging in C57BL/6N mouse.&#160;In vivo 3D imaging of pancreas (green) of C57BL/6N mouse intravenously injected with an anti-CD31 antibody conjugated with the Alexa 647 fluorophore (red). Scale bar is depicted in the video. This video corresponds to Figure 3C,D. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/SupplVideo_1.mp4" target="_blank">Please click here to download this Video.</a>
Supplementary video 2: In vivo 3D pancreatic cancer (PANC-1 NucLight Red) imaging in BALB/C Nude mouse.&#160;In vivo 3D imaging of pancreatic cancer (red) implanted in BALB/C Nude mouse intravenously injected with an anti-CD31 antibody conjugated with the Alexa 647 fluorophore (blue). Scale bar is depicted in the video. This video corresponds to Figure 7C,D. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62538/SupplVideo_2.mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

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.

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 ...

stabilized-longitudinal-vivo-cellular-level-visualization-pancreas

Nanyang Technological University

Research

JoVE Journal

Medicine

4.0K Views.  Seoul National University Bundang Hospital. Dit protocol beschrijft gestabiliseerde en repetitieve cellulaire in vivo beeldvorming van de alvleesklier met een nieuw pancreas intravital imaging venster. Het belangrijkste voordeel van deze techniek is dat het mogelijk is om bewegingsloze, driedimensionale beeldvorming uit te voeren met resolutie tot het cellulaire niveau gedurende drie weken in de alvleesklier van een levende muis. Begin met het voorbereiden van het operatieplatform en het desinfecteren van de oppervlakken met 70% ethano...

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

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

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 ...

Cancer Research

Murine Pancreatic Imaging Using Implanted Stabilized Window

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 

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 ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

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.remotexs.ntu.edu.sg/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 ...

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Rodent Models

Adult

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

Source: Park, I. and Kim, P. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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).
In this video, we describe the intravital imaging of a mouse pancreas through a stabilized window for long-term visualization of cellular dynamics.

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

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

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