Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Summary

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Abstract

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ ≤ 30 µm in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Introduction

Disturbances in retinal perfusion have been implicated in the pathogenesis of various ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and glaucoma^1,2,3. Thus, studies aimed at measuring vascular reactivity in the retina are important to understand the pathophysiology of these diseases and to develop new treatment approaches.

Due to the possibility of gene manipulation in the murine genome, the mouse has become a widely used animal model for studies of the cardiovascular system⁴. However, because of t....

Protocol

The experimental procedures of this study were approved by the Animal Care Committee of Rhineland-Palatinate, Germany. Animal care conformed to the institutional guidelines and The Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. Animals were treated according to the EU Directive 2010/63/EU for animal experiments. Male C57BL/6J mice (The Jackson Laboratory, Bar Harbour, ME, USA) aged 3-4 months were used for the experiments. Animals were housed under standard conditions (temperature 23 ± 2 °C, humidity range 55 ± 10% and 12 h light/dark cycles), and had access to standard mou....

Results

U-46619 produced concentration-dependent vasoconstrictor responses in retinal arterioles from wild-type mice of the C57Bl/6J background. At a concentration of 10^-6 M, reduction in luminal diameter was ≈50% from resting diameter. Figure 9A shows a representative concentration-response curve of one retinal arteriole. In arterioles preconstricted with U46619, cumulative administration of acetylcholine evoked concentration-dependent increases in lumina.......

Discussion

The measurement of vascular responses in the mouse retina is challenging due to the small size of retinal blood vessels. With the presented technique, retinal arterioles are visualized by transmitted light microscopy. This is possible, because the isolated retina is translucent. The advantage of the technique is the high optical resolution. The calculated spatial resolution is 11 px/µm. However, the real resolution for this optical system that uses white light is between 200 and 300 nm, which is explained by the Abb.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Steel Scissors	Carl Roth GmbH	3576.1	1x 140 mm
Eye Scissors	Geuder	G-19390	1x straight, 10.5 cm
Precision tweezers, straight with fine tips	Carl Roth GmbH	LH68.1	2x type 4
Precision tweezers, straight with extra fine tips	Carl Roth GmbH	LH53.1	2x type 5
Vannas capsulotomy scissors	Geuder	19760	1x straight, 77 mm
Student Vannas Spring Scissors	Fine Science Tools	91501-09	1x curved,
Barraquer Needle Holder	Geuder	G-17500	1x curved, 120 mm
Needle	Becton, Dickinson and Company	305128	1x 30 G
Glass Capillaries (for producing micropipettes)	Drummond Scientific Company	9-000-1211	1x (1.2 x 0.8 mm; outer/inner diameter)
Nylon Suture	Alcon	198001	1x 10-0
Nunclon cell culture dish	Thermo Fisher Scientific	153066	1x 35 mm diameter
Nunclon cell culture dish	Thermo Fisher Scientific	172931	1x 100 mm diameter
Discofix C	Braun	16500C	10 cm
Histoacryl adhesive	B. Braun Surgical, S.A.	1050052
Name	Company	Catalog Number	Comments
Equipment
Pericyclic pump  (CYCLO II)	Carl Roth GmbH	EP76.1	1x
Vertical Pipette Puller Model 700C	David Kopf Instruments		1x
Microscope (Vanox-T AH-2)	Olympus		1x
Water immersion objective LUMPlanFL, 1.0 NA	Olympus		1x
Digital camera (TK-C1381)	JVC		1x
Perfusion chamber	self-made		1x
Name	Company	Catalog Number	Comments
Drugs and Solutions
Ethanol	Carl Roth GmbH	K928.4
Calcium chloride dihydrate (CaCl2)	Carl Roth GmbH	5239.1
Kalium chloride (KCl)	Carl Roth GmbH	6781.1
Kalium dihydrogen phosphate (KH2PO4)	Carl Roth GmbH	3904.2
Magnesium sulphate (MgSO4)	Carl Roth GmbH	261.2
Sodium chloride (NaCl)	Carl Roth GmbH	9265.2
Sodium hydrogen carbonate (NaHCO3)	Carl Roth GmbH	0965.3
α-(D)-(+)- Glucose monohydrate	Carl Roth GmbH	6780.1
9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619)	Cayman Chemical	16450
Acetylcholine chloride	Sigma-Aldrich	A6625-25G