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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

All the procedures described here were performed with prior approval by the Dalhousie University Committee on Laboratory Animals (UCLA).

1. Preparation

  1. Lung imaging system: To prepare the window, administer a thin layer of vacuum grease to the top of the outer ring while avoiding contamination of the vacuum channel. Place a clean 8 mm glass coverslip on the window and gently press down to create a seal.
  2. Widefield Fluorescence Microscope: Perform imaging w.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

The authors would like to thank Dr. Pina Colarusso, who provided significant expertise in the editing and revising of this manuscript.

....

Materials

NameCompanyCatalog NumberComments
1 mL BD Luer Slip Tip Syringe sterile, single useBecton, Dickinson and Company3096591 mL syringe
ADSON Dressing Forceps, Tip width 0.6 mm, teeth length 11.5 mm, 12 cmRWD Life Science Co.F12002-12Blunt forceps
Albumin-Fluorescein IsothiocyanateSigma-AldrichA9771-1GFITC-albumin
Alcohol Swab Isopropyl Alcohol 70% v/vCanadian Custom Packaging Company80002455Alcohol wipe
AVDC110 Advanced Digital Video ConverterCanopus00631069602029Digital video converter
B/W - CCD - CameraHorn ImagingBC-71Camera
Bovie Deluxe High Temperature Cautery KitFine Science Tools18010-00Cauterizer
C57BL/6 MiceCharles River Laboratories InternationalC57BL/6NCrlC57BL/6 Mice
Cotton Tipped ApplicatorsPuritan806-WCCotton applicator
CS-8R 8mm Round Glass CoverslipWarner Instruments64-0701Glass coverslip
Digital Pressure GaugeITM Instruments Inc.DG2551L0NAM02L0IM&VDigital Pressure Gauge
Dr Mom Slimline Stainless LED OtoscopeDr. Mom Otoscopes1001Otoscope
Ethyl Alchohol 95% VolCommercial AlcoholsP016EA9595% ethanol
Fine Scissors - Martensitic Stainless SteelFine Science Tools14094-11Scissors
Fisherbrand Colored Labeling TapeFisher Scientific1590110Labeling tape
Gast DOA-P704-AA High-Capacity Vacuum PumpCole-Parmer Canada CompanyZA-07061-40Vacuum pump
Hartman HemostatsFine Science Tools13003-10Hemostatic forceps
High Vacuum GreaseDow CorningDC976VFVacuum grease
Isoflurane USPFresenius KabiCP0406V2Isoflurane
LIDOcaine HCl Injection 1% 50 mg/5 mLTeligent Canada0121AD01Lidocaine HCl 1%
Lung SurgiBoardLuxidea, Inc.IMCH-0001Designed for intravital microscopy of the lung
Mineral OilTeva Canada00485802Mineral oil
Mouse Endotracheal Intubation KitKent Scientific CorporationETI-MSEIntubation stand, anesthesia mask, 20 G endotracheal cannula, fibre optic cable
MST49 Fluorescence MicroscopeLeica Microsystems10 450 022Fluorescence Microscope
N Plan L 20x/0.40 Long Working Distance Microscope ObjectiveLeica Microsystems56603520x objective
Non-Woven Sponges 2" x 2"AMD-RitmedA2101-CHGauze
Optixcare Eye Lube PlusAventix5914322Tear gel
Original Prusa i3 MK3S+ 3D PrinterPrusa ResearchPRI-MK3S-KIT-ORG-PEI3D printer
Oxygen, CompressedLinde Canada Inc.Oxygen
PrecisionGlide Needle 30 G x 1/2 (0.3 mm x 13 mm)Becton, Dickinson and Company30510630 G needle
Pyrex 5340-2L 5340 Filtering Flasks, 2000 mLCole-Parmer Canada Company5340-2LVacuum flask
Rhodamine 6 GSigma-Aldrich252433Rhodamine 6G
Secure Soft Cloth Medical Tape - 3"PrimedPM5-630709Cloth tape
Silastic Medical Grade Tubing .040 in. ID x .085 in. ODDow Corning602-2051.0 mm I.D. polyethylene tubing
Somnosuite Low-Flow Anesthesia SystemKent Scientific CorporationSS-01, SS-04-moduleSmall rodent ventilator, Low-flow anesthesia system, Heating pad, Rectal temperature probe, Pulse oximeter
Tissue Forceps, 12.5cm long, Curved, 1 x 2 TeethWorld Precision Instruments501216Toothed forceps
Transpore Medical Tape, 1527-1, 1 in x 10 yd (2.5 cm x 9.1 m)3M7000002795Medical tape
Tubing,Clear,3/8 in Inside Dia.Grainger CanadaUSSZUSA-HT33141.0 cm I.D. polyethylene tubing
Whatman 6720-5002 50 mm In-Line Filters, PTFE, 0.2 µmCole-Parmer Canada Company6720-5002Inline 0.2µm filter

References

  1. Alizadeh-Tabrizi, N., Hall, S., Lehmann, C. Intravital imaging of pulmonary immune response in inflammation and infection. Frontiers in Cell and Developmental Biology. 8, 620471 (2021).
  2. Gaertner, M., et al.

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