Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

Summary

We demonstrate a method to label the walls of the retinal vasculature and adherent leukocytes. These adherent leukocytes can then be counted under a fluorescence microscope as a parameter of inflammation or the response of that inflammation to therapies.

Abstract

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels is characteristic of inflammation and has been causally linked to capillary occlusion in a variety of tissues and diseases, including diabetic retinopathy.

Leukostasis has been reported for years as a life-threatening complication of hyperleukocytosis and can only be diagnosed clinically. Given the importance of the phenomenon, intensive research has been done to understand the potential mechanism(s) that lead to its manifestation; however, there is no gold-standard technique in laboratory settings to visualize and quantify the severity of the event.

In the method summarized below, the vasculature is initially perfused with a buffer to remove blood, and then, concanavalin A is perfused into the vasculature where it binds to all exposed cell walls and causes especially bright staining of leukocytes. If the perfusion to remove all unbound blood cells was successful, the remaining fluorescently labeled leukocytes are bound to the vasculature, and they can be manually quantified using any available fluorescence microscope.

Introduction

Leukocytes (white blood cells, WBCs) play an important role in the optimal function of the vasculature such as maintenance of the blood fluidity and regulation of thrombus resolution¹. They also play a key role in some pathological conditions, such as adhering to the luminal wall of the vasculature for prolonged periods of time leading to vessel obstruction, at least temporarily, a phenomenon known as leukostasis^2,3.

Diabetic retinopathy is one of the most common complications of long-term diabetes and one of the leading cau....

Protocol

The protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California Irvine and conforms to governmental regulations regarding the care and use of laboratory animals. There are no stop points in this protocol. The average time per mouse is 30 min.

1. Preparing the perfusion stage

1. Warm up the 0.9% saline bag and concanavalin A solution in a 37 ^oC water bath for 20-30 min before use.
   .......

Discussion

Leukostasis in humans refers to symptoms and clinical findings associated with hyperleukocytosis (total leukocytes (WBCs) count >100,000/µL) and is a medical emergency²⁰. The mechanism(s) that lead to leukostasis are under intensive research. To date, the study of leukostasis in humans in vivo is not yet possible and researchers need to rely on animal models to understand this process. Different diseases present leukostasis and having a detailed protocol to visualize the phenomen.......

Materials

Name	Company	Catalog Number	Comments
10 mL syringe
4-way stopcock Luer lock I.V. line valve	Baxter	2C6204
Concanavalin A solution	Vector	FL-1001	Prepare in PBS 1 mg/mL
Dissecting tools set			Includes hemostats, scissors and forceps
FIJI			Software for image processing
Fluorescence microscope	Nikon	Eclipse Ni
Forceps, Dumont #5, Biological grade tip	Electron Microscopy Sciences (EMS)	72700-D
Gavage Needle 1.25 mm OD barrel tip x 30 mm	Fine Science	18060-20
Halstead Mosquito Forceps	Fisher Scientific	13-812-10
I.V. Catheter set with regulating clamp 70 inches	Baxter	2C5417s
I.V. Pole
Lint free tissue			Kimpwipes is an option
Micro dissecting spring scissors, Vannas, 3 mm straight	ROBOZ	RS-5620
Micro spatula	Fine Science Tools (FST)	10091-12
Nikon		NIS-Elements (AR 5.30.03 64-bit)	Software for image acquisition
Petri dish (100 mmx15 mm)	Corning	351029
Phosphate buffered saline (PBS)
Pink dental wax	Electron Microscopy Sciences (EMS)	72670
Pressure infuser	Infusurge	4010
Razor blades, GEM single edge stainless steel, Teflon coated	Electron Microscopy Sciences (EMS)	71970
Saline 0.9%, veterinary grade, 1000 mL	Baxter	04925-04-10
Small dissecting scissors, curved blunt end 22 mm	ROBOZ	RS 5983