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Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.
Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.
Intravital microscopy (IVM) is a useful imaging tool for visualizing and studying various biophysical processes in vivo. The lung is highly challenging to image in vivo due to its enclosed location, the fragile nature of its tissue, and motion artifacts induced by respiration and heartbeat1,2. Various intravital microscopy (IVM) setups have been developed for real-time imaging of leukocyte-endothelial interactions in pulmonary microcirculation to overcome these challenges. Such approaches are based on surgically exposing and stabilizing the lung for imaging.
Animal....
All the procedures described here were performed with prior approval by the Dalhousie University Committee on Laboratory Animals (UCLA).
1. Preparation
To illustrate results achievable through this protocol, acute lung injury (ALI) was induced 6 h prior to imaging using a model of intranasal bacterial lipopolysaccharide (LPS) instillation. Briefly, mice (n = 3) were anesthetized with isoflurane, and small droplets of LPS from Pseudomonas aeruginosa in sterile saline (10 mg/mL) were pipetted into the left naris at a dosage of 5 mg/kg. This was compared to naïve mice (n = 3; no intranasal administration).
Upon imaging, a successfu.......
The protocol presented here requires practice and attention to a few critical steps. First, it is important to prepare the imaging window prior to initiating intubation and surgery. Use a minimal amount of vacuum grease to coat the outer ring of the imaging window, apply the cover glass, and test suction with a drop of distilled water. Preparing this in advance will prevent the exposed lung from drying out during the setup otherwise. While it is possible to flush with warm saline, doing so may risk damaging the fragile p.......
The authors would like to thank Dr. Pina Colarusso, who provided significant expertise in the editing and revising of this manuscript.
....Name | Company | Catalog Number | Comments |
1 mL BD Luer Slip Tip Syringe sterile, single use | Becton, Dickinson and Company | 309659 | 1 mL syringe |
ADSON Dressing Forceps, Tip width 0.6 mm, teeth length 11.5 mm, 12 cm | RWD Life Science Co. | F12002-12 | Blunt forceps |
Albumin-Fluorescein Isothiocyanate | Sigma-Aldrich | A9771-1G | FITC-albumin |
Alcohol Swab Isopropyl Alcohol 70% v/v | Canadian Custom Packaging Company | 80002455 | Alcohol wipe |
AVDC110 Advanced Digital Video Converter | Canopus | 00631069602029 | Digital video converter |
B/W - CCD - Camera | Horn Imaging | BC-71 | Camera |
Bovie Deluxe High Temperature Cautery Kit | Fine Science Tools | 18010-00 | Cauterizer |
C57BL/6 Mice | Charles River Laboratories International | C57BL/6NCrl | C57BL/6 Mice |
Cotton Tipped Applicators | Puritan | 806-WC | Cotton applicator |
CS-8R 8mm Round Glass Coverslip | Warner Instruments | 64-0701 | Glass coverslip |
Digital Pressure Gauge | ITM Instruments Inc. | DG2551L0NAM02L0IM&V | Digital Pressure Gauge |
Dr Mom Slimline Stainless LED Otoscope | Dr. Mom Otoscopes | 1001 | Otoscope |
Ethyl Alchohol 95% Vol | Commercial Alcohols | P016EA95 | 95% ethanol |
Fine Scissors - Martensitic Stainless Steel | Fine Science Tools | 14094-11 | Scissors |
Fisherbrand Colored Labeling Tape | Fisher Scientific | 1590110 | Labeling tape |
Gast DOA-P704-AA High-Capacity Vacuum Pump | Cole-Parmer Canada Company | ZA-07061-40 | Vacuum pump |
Hartman Hemostats | Fine Science Tools | 13003-10 | Hemostatic forceps |
High Vacuum Grease | Dow Corning | DC976VF | Vacuum grease |
Isoflurane USP | Fresenius Kabi | CP0406V2 | Isoflurane |
LIDOcaine HCl Injection 1% 50 mg/5 mL | Teligent Canada | 0121AD01 | Lidocaine HCl 1% |
Lung SurgiBoard | Luxidea, Inc. | IMCH-0001 | Designed for intravital microscopy of the lung |
Mineral Oil | Teva Canada | 00485802 | Mineral oil |
Mouse Endotracheal Intubation Kit | Kent Scientific Corporation | ETI-MSE | Intubation stand, anesthesia mask, 20 G endotracheal cannula, fibre optic cable |
MST49 Fluorescence Microscope | Leica Microsystems | 10 450 022 | Fluorescence Microscope |
N Plan L 20x/0.40 Long Working Distance Microscope Objective | Leica Microsystems | 566035 | 20x objective |
Non-Woven Sponges 2" x 2" | AMD-Ritmed | A2101-CH | Gauze |
Optixcare Eye Lube Plus | Aventix | 5914322 | Tear gel |
Original Prusa i3 MK3S+ 3D Printer | Prusa Research | PRI-MK3S-KIT-ORG-PEI | 3D printer |
Oxygen, Compressed | Linde Canada Inc. | Oxygen | |
PrecisionGlide Needle 30 G x 1/2 (0.3 mm x 13 mm) | Becton, Dickinson and Company | 305106 | 30 G needle |
Pyrex 5340-2L 5340 Filtering Flasks, 2000 mL | Cole-Parmer Canada Company | 5340-2L | Vacuum flask |
Rhodamine 6 G | Sigma-Aldrich | 252433 | Rhodamine 6G |
Secure Soft Cloth Medical Tape - 3" | Primed | PM5-630709 | Cloth tape |
Silastic Medical Grade Tubing .040 in. ID x .085 in. OD | Dow Corning | 602-205 | 1.0 mm I.D. polyethylene tubing |
Somnosuite Low-Flow Anesthesia System | Kent Scientific Corporation | SS-01, SS-04-module | Small rodent ventilator, Low-flow anesthesia system, Heating pad, Rectal temperature probe, Pulse oximeter |
Tissue Forceps, 12.5cm long, Curved, 1 x 2 Teeth | World Precision Instruments | 501216 | Toothed forceps |
Transpore Medical Tape, 1527-1, 1 in x 10 yd (2.5 cm x 9.1 m) | 3M | 7000002795 | Medical tape |
Tubing,Clear,3/8 in Inside Dia. | Grainger Canada | USSZUSA-HT3314 | 1.0 cm I.D. polyethylene tubing |
Whatman 6720-5002 50 mm In-Line Filters, PTFE, 0.2 µm | Cole-Parmer Canada Company | 6720-5002 | Inline 0.2µm filter |
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