A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Summary

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Abstract

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Introduction

The retina is a neurosensory tissue for visual function. Since a substantial amount of oxygen is needed for visual function, the retina is known as one of the highest oxygen demanding tissues in the body¹. The retina is susceptible to vascular diseases as oxygen is delivered through blood vessels. Various types of vascular diseases, such as diabetic retinopathy and retinal blood vessel (veins or arteries) occlusion, can induce retinal ischemia. To investigate pathological mechanisms of retinal ischemia, reproducible and clinically relevant experimental models of retinal ischemia are considered necessary. Middle cerebral artery occlusion (MCAO) ....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Keio University School of Medicine.

1. Preparation of surgical instruments and animals

1. Autoclave surgical instruments and keep them in 70% ethyl alcohol. Prior to each new surgical procedure, clean surgical instruments carefully using 70% ethyl alcohol.
2. Prepare male BALB/cAJc1 mice (6 weeks old, 26-28 kg) in a specific-pathogen-free (SPF) room to maintain sterile conditions before, during and after the surgery.

2. Transient bilateral common carotid artery occlusion (tBCCAO)

Results

After systemic circulation of FITC-dextran for 2 minutes, retinal vasculatures of the left and right retinas in the sham-operated mice and tBCCAO-operated mice were examined (Supplementary Figure 1). FITC-dextran was fully visible in the both retinas in the sham-operated mice and the left retina in the tBCCAO-operated mice, while it was partially detectible in the right retina in the tBCCAO-operated mice.

After tBCCAO, eyelid drooping was examined (Figure .......

Discussion

In the study, we have shown that tBCCAO, using simple sutures and a clamp, could induce retinal ischemia and accompanying retinal dysfunction. Furthermore, we have demonstrated our current protocol for the development of a mouse model of retinal ischemia is easier and faster in comparison with other previous protocols for the development of retinal ischemic injury models^2,3,7.

Anatomically, the left a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Atipamezole hydrochloride	Zenoaq	Antisedan	For anti-anesthesia
Applied Biosystems 7500 Fast	Applied Biosystems	-	For qPCR
Butorphanol tartrate	Meiji Seika Pharma	Vetorphale	For anesthesia
BZ-II Analyzer	KEYENCE	-	For an image merge
BALB/cAJc1	CLEA	-	Mouse strain
β-Actin (8H10D10) Mouse mAb	CST	3700	For western blot
Clamp Forcep	World Precision Instruments	WPI 500451	For surgery
Dumont forceps #5	Fine Science Tools	11251-10	For surgery
DAPI solution	Dojindo	340-07971	For IHC
Envisu SD-OCT system	Leica	R4310	For OCT
FITC-dextran	Merk	FD2000S	For retinal blood perfusion
Fluorescence microscope	KEYENCE	BZ-9000	For fluorescence detection
Gatifloxacin hydrate	Senju Pharmaceutical	Gachifuro	For anti-bacterial infection
GFAP Monoclonal Antibody (2.2B10)	Thermo	13-0300	For IHC
Heating pad	Marukan	RH-200	For surgery
HIF-1α (D1S7W) XP Rabbit mAb	CST	36169	For western blot
ImageQuant LAS 4000 mini	GE Healthcare	-	For chemiluminescence
Midazolam	Sandoz K.K	SANDOZ	For anesthesia
Microtome Tissue-Tek TEC 6	Sakura	-	For sectioning
Medetomidine	Orion Corporation	Domitor	For anesthesia
Needle holder	Handaya	HS-2307	For surgery
PuREC	MAYO Corporation	-	For ERG
Scissor	Fine Science Tools	91460-11	For surgery
Sodium hyaluronate	Santen Pharmaceutical	Hyalein	For eye lubrication
Tropicamide/Penylephrine hydrochloride	Santen Pharmaceutical	Mydrin-P	For mydriasis
6-0 silk suture	Natsume	E12-60N2	For surgery