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Podsumowanie

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

Abstract

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.

Introduction

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 field^1,2. 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 diagnosis^3,4, 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 vivo^7,8 (e.g., slicing or digestion of the pancreas), non-physiologic⁹ (e.g., exteriorization of the pancreas), and heterotopic conditions^10,11,12 (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 microscopy^19,20 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 studies^{10,18,19,20,21,22,23}.

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.

Protokół

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 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. 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. 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. 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. Sterilize all surgical tools, cover glass (with PEG or not), and imaging windows using an autoclave. Apply PEG coating to the cover glass to prevent inflammatory response and increase biocompatibility, which is suitable for long-term imaging. 2. Surgery Prepare a sterile surgical platform and sterilize the surfaces with 70% ethanol. NOTE: For longitudinal imaging sessions, aseptic technique is essential. 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. Monitor the body temperature using a rectal probe with a homeothermic controlled heating pad. Shave the left flank of the mouse and apply three rounds of alternating alcohol and iodine-based scrub. NOTE: If depilatory cream is used, do not leave longer than 1 minute to avoid chemical burn. Make a 1.5 cm incision on the left flank of the mouse and dissect the skin and muscle. Perform a purse-string suture with a black or nylon 4-0 suture in the incision margin. Use a micro retractor on the incision and gently expose the spleen. Carefully pool the spleen with ring forceps and identify the pancreas. Place the window at the flank of the mouse and pass the spleen and pancreas through the open space of the window. Manipulation of the pancreas has to be very gentile as it can cause bleeding or pancreatitis Gently place the pancreas on the plate of the imaging window; the spleen will be placed on the open space of the window. 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. Although maximal injection volume is uncertain, decrease the volume as possible to avoid the pressure effect in the pancreas. Apply drops of N-butyl cyanoacrylate glue on the margin of the imaging window. 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. Gently apply a 12 mm round cover glass to the margin of the imaging window. Pull the suture loop to fit into the lateral groove of the window and tie it three times. Cut the maximal proximal site of the knot to prevent the interruption of tight stitches when these mice are awake. 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 ketoprofen should be considered every 24 hours for 1-3 days. ​NOTE: Analgesia influences insulin secretion in response to glucose9. The choice and timing of analgesia must be individualized for the experimental purpose. 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. 3. Intravital imaging Turn on the intravital microscope including the laser power. Turn on the heating pad and set the homeothermic regulation to 37 °C. NOTE: Alternatively, use a passive heating pad or lamp with frequent control if there is no homeothermic regulation. 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. Insert a vascular catheter for the injection. 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. Sterilize the tail vein with a 70% ethanol spray. Insert a 30 G catheter into the lateral tail vein. Regurgitation of blood will be visualized in the PE10 tube. Apply a silk tape on the catheter to stabilize it. Inject FITC/TMR dextran or other fluorescent probes (25 µg 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. Transfer the mouse from the surgical platform to the imaging stage. Insert a rectal probe to automatically control the body temperature with the homeothermic heating pad system. 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. Perform intravital imaging. 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 µm). 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 µm or 250 x 250 µm). In this experiment, the lateral and axial resolution was approximately 0.5 µm and 3 µm, respectively. 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.

Representative Results

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 sa…

Discussion

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 significantl…

Materials

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