In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Summary

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Abstract

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

Introduction

The endothelial glycocalyx is an extracellular layer of proteoglycans and associated glycosaminoglycans lining the vascular intima. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that regulates a variety of endothelial functions including fluid permeability¹, neutrophil-endothelial adhesion², and the mechanotransduction of fluid shear stress³.

Historically, the glycocalyx has been underappreciated due to its aberrance in cultured cell preparations^{4, 5} and its degradation during standard tissue fixation and processing⁶.....

Protocol

1. Preparation of Surgical Tubing, Vascular Catheters, Chest Wall Window

1. Intravital microscopy stage. We custom-made a plexiglass stage upon which the anesthetized mouse lies during microscopy. This stage accommodates both a 15 cm by 10 cm flexible plastic cutting board (upon which the mouse lies during induction of anesthesia, tracheostomy placement, and venous catheterization) as well as a similarly-sized heating element (located underneath the cutting board).
2. Mouse thoracostomy tube preparation (Figure 1). A 10 cm length of PE 50 tubing (Intramedic, inner diameter 0.58 mm, outer diameter 0.965....

Results

The experimental approach described in steps 1-6 will allow capture of multiple frames of simultaneous DIC (brightfield) and fluorescent images. To determine ESL thickness, recorded images are reviewed by a blinded observer after completion of the experimental protocol. Using an in-focus frame, subpleural microvessels (< 20 μm diameter) are identified; at least 3 microvessels are typically found on a single frame (Figure 10). Using image analysis software (NIS Elements, Nikon), vascular width.......

Discussion

Coincident with the expanding use of in vivo microscopy, there is increasing appreciation for both the substantial size of the ESL as well as its numerous contributions to vascular function. These emerging data, however, are primarily derived from studies of the systemic vasculature. Indeed, use of in vivo microscopy in the lung is technically challenging, given significant pulmonary and cardiac motion artifact.

Several recent technical advances have allowed for stabi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of Reagent
FITC-dextran (150 kDa)	Sigma	FD150S
TRITC-dextran (150 kDa)	Sigma	T1287
Streptavidin-coated fluorescent microspheres	Bangs Laboratories	CP01F/10428	Dragon Green fluorescence (similar to FITC)
Ketamine	Moore Medical
Xylazine	Moore Medical
Anti-ICAM-1 biotinylated antibody	eBioscience	Clone YN1/1.7.4	1:50 dilution
Isotype biotinylated antibody	eBioscience	IgG2b eB149/10H5	1:50 dilution
EQUIPMENT
Mechanical ventilator	Harvard Apparatus	Inspira
Tracheostomy catheter	Harvard Apparatus	730028
Electrocautery apparatus	DRE Medical	Valleylab SSE-2L
Bipolar cautery forceps	Olsen Medical	10-1200I	9.9cm McPherson
Temperature control system	World Precision Instruments	ATC1000
Syringe pump	Harvard Apparatus	Pump 11 Elite
Microscope (widefield)	Nikon	LV-150
Microscope (confocal)	Nikon	A1R
Image splitter	Photometrics	DV2
CCD camera	Photometrics	CoolSNAP HQ2
Image processing software	Nikon	NIS Elements
Polyvinylidene membrane	Kure Wrap
Circular cover slip	Bellco	5CIR-1-BEL	5 mm, #1 thickness
Glue (cover slip to membrane)	Pattex	Flussig (liquid)	For affixing cover slip to membrane
Glue (cover slip to mouse)	Pattex	Gel	For attaching membrane to mouse
Surgical tubing	Intramedic	PE50, PE10
Suture	Fisher		4:0 silk
Electric razor	Oster	78997
Curved surgical forceps	Roboz
Straight surgical forceps	Roboz
Surgical scissors	Roboz
Surgical microscissors	Roboz
Surgical needle driver	Roboz
Surgical tape	Fisher
Kitchen sponges (cut into wedges)	various