Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

Summary

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

Abstract

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Introduction

Different models have been presented to study retinal diseases, including both in vivo and in vitro models. The usage of animals in research is still a matter of continuous ethical and translational debate¹. Animal models involving rodents such as mice or rats are commonly used in retinal research^2,3,4. However, clinical concerns have arisen because of the different physiological functions of the retina in rodents compared to humans, such as the absence of the macula or differences in color vision⁵. The usage o....

Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Oakland University, Rochester, MI, USA and followed the guidelines established by the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Animal preparation

1. Keep the animals housed under a constant temperature in a light-controlled environment. The temperature settings for the animal housin.......

Discussion

Our lab has been studying the pathophysiological changes that promote retinal microvascular dysfunction for years^{31,32,33,34,35,36}. Retinal explants are one of the techniques that can be of great value to use as a model for studying retinal diseases such as diabetic retinopathy or degenerative retinal diseases. Having a contr.......

Materials

Name	Company	Catalog Number	Comments
Adult C57Bl/6J mice	The Jackson Laboratory, Bar Harbor, ME, 04609, USA	664
All-in-One Fluorescence Microscope	KEYENCE CORPORATION OF AMERICA, IL, 60143, U.S.A.	BZ-X800
B27 supplements	Thermo scientific. Waltham, MA, 02451, USA	Gibco #17504-04
Blockade blocking solution	Thermo scientific. Waltham, MA, 02451, USA	B10710
DMEM F12	Thermo scientific. Waltham, MA, 02451, USA	Gibco #11320033
Goat anti-Rabbit IgG.	Thermo scientific. Waltham, MA, 02451, USA	F-2765
GSL I, BSL I (Isolectin)	Vector Laboratories. Burlingame, CA 94010,USA	B-1105-2
Hanks Ballanced Salt Solution (HBSS)	Thermo scientific. Waltham, MA, 02451, USA	Gibco #14175095
Micro Scissors, 12 cm, Diamond Coated Blades	World Precision Instruments,FL 34240, USA	Straight (503365)
N2 supplements	Thermo scientific. Waltham, MA, 02451, USA	Gibco #17502-048
Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates	Thermo scientific. Waltham, MA, 02451, USA	140652
Paraformaldehyde 4% in PBS	BBP, Ashland, MA, 01721 USA	C25N107
Penicillin-Streptomycin (10,000 U/mL)	Thermo scientific. Waltham, MA, 02451, USA	15140148
PROLONG DIAMOND ANTIFADE 4′,6-diamidino-2-phenylindole (DAPI).	Thermo scientific. Waltham, MA, 02451, USA	P36962
Rabbit Anti-NeuN Antibody	Abcam.,Cambridge, UK	ab177487
Rabbit Glial Fibrillary Acidic Protein (GFAP) Antibody	Dako,Carpinteria, CA 93013, USA.	Z0334
Texas Red	Vector Laboratories. Burlingame, CA 94010,USA	SA-5006-1
TritonX	BioRad Hercules, CA,  94547,USA	1610407