Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Summary

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Abstract

Trypsin digest is the gold standard method to analyze the retinal vasculature ^1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations ^6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Introduction

Visualizing the vasculature of the retina is an extremely important approach to dissect the mechanisms of various eye diseases such as diabetic retinopathy. It allows one to assess the earliest vascular abnormalities, including microaneurysms, capillary degeneration, and pericyte loss ^8,9 . To date, there have been several techniques developed to analyze the retinal vasculature. Perfusion of various dyes has been used to highlight the vessels, but all have shared similar limitations. Injection rarely highlights the entire retinal vasculature unless given at a high pressure, which risks rupturing and damaging the vessels ¹. Immunostaining of vascu....

Protocol

1. Retinal Preparation

1. Enucleate the mouse eye. Using one hand, open the eye lids so that the eye is visible. With the other hand, using a curved forceps (curved facing upwards), apply pressure on superior and inferior aspects of the orbit until the globe protrudes. Gently close forceps at the posterior aspect of the eye and lift in a continuous motion to enucleate the eye.
2. Fix eye with 10% neutral buffered formalin for at least 24 hr.
3. Place eye in PBS solution in a small Petri dish.
4. Dissect out retina carefully under a microscope, taking care to avoid inducing large tears. Make an initial cut in the cornea with dissection sci....

Results

The final product of a successful procedure is a flat-mount of the entire network of the mouse retinal vasculature, with the architecture maintained, stained with either PAS/hematoxylin or H&E, as shown in Figures 2-4. Clear differentiation of endothelial cells and pericytes can be seen as shown in Figure 3. In the retina, the nuclei of endothelial cells are oval or elongated and lie entirely within the vessel wall. Pericyte nuclei are small, spherical, stain densely and generally ha.......

Discussion

Trypsin digest is a standard method to assess the vasculature of the retina. Unfortunately, it is technically challenging and can result in high rate of specimen loss if not performed correctly. Furthermore, the procedure is especially difficult in mice, which can limit the application of this technique in the commonly used genetic animal models of eye diseases. This paper provides guidance on how to perform the procedure effectively and consistently in mouse eyes.

There are several critical s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
10% neutral buffered formalin	Fischer Scientific	SF100-4
Trypsin (1:250)	Amresco	0458-50G	Make 3% trypsin in 0.1 M Tris
TRIZMA base	Sigma	T1503-1KG	Create 0.1 M Tris Buffer (pH 7.8 @ 37 °C)
TRIZMA	Sigma	T3253-500G
Steritop sterile vacuum bottle	Millipore	SCGPS05RE	Create filtered water
EQUIPMENT
Dissecting microscope			Any microscope that allows good visualization of the retina is adequate. (10x is sufficient with working distance of 24 mm)
Straight scissors	Fine Science Tools	15024-10	Cutting edge: 8 mm; Tip diameter: 0.2 mm; 10 cm
Straight Forceps (Inox)	Dumont #5	11252-20	Biologie tip; 0.05 x 0.02 mm; 11 cm
Curved Forcepts (Dumostar)	Dumont #7	11297-10	Biologie tip; 0.07 x 0.04 mm; 11.5 cm
Dubnoff Metabolic Shaker Incubator	Precision Scientific	BDG59442	Shaker optional
24-well dish	Falcon	353047
Microscope Slides	VWR	82027-132