In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Summary

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Abstract

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1^gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases^1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1^gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 µm and 6 µm, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies⁷, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate⁸.

Introduction

In experimental ophthalmology, examination of retinal pathology is usually evaluated using histological techniques. However, histology requires animal euthanization and may cause alteration to the actual properties of the tissue. SD-OCT and SLO are routinely used in clinical ophthalmology for diagnostic purposes and for the monitoring of several retinal diseases such as diabetic macular edema⁹, anterior ischemic optic neuropathy¹⁰, or retinitis pigmentosa¹¹. SD-OCT and SLO are non-invasive techniques that generate high-resolution images of the retina, which are visualized through the dilated pupil....

Protocol

In all procedures, BALB/c adult male and female mice who express gfp under the promoter of Cx3cr1 were used²⁴. Mice were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and all procedures were approved from the Swiss government according to the Federal Swiss Regulations on Animal Welfare. Mice were anesthetized by a subcutaneous injection of medetomidine hydrochloride (0.75 mg/kg) and ketamine (45 mg/kg). Proper anesthesia was confirmed by monitoring the respiratory rate and the animal's reflex against a tail pinch. At the end of the experiments, mice were euthanized with CO₂

Results

Using the protocol presented here, SD-OCT scans and SLO images were obtained from Cx3cr1^gfp/gfp mice in the same imaging session. Figure 3 includes representative SD-OCT single scans obtained with a 30 ° or a 55 ° lens (Figure 3A) and representative SLO images obtained with a 55 ° or a 102 ° lens, where gfp positive microglia cells are visualized. Higher reflectivity of the choroid is observed in the SD-OCT sc.......

Discussion

The present article demonstrates a protocol for the acquisition of retinal B-scans and imaging of gfp positive microglia distribution in the mouse retina in the same imaging session. SD-OCT and SLO are increasingly used in animal models of retinal disease to provide information of retinal alterations over time^{10,14,17,18,21}. With this protocol, Cx3cr1^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Spectralis Imaging system (HRA+OCT)	Heidelberg Engineering, Germany	N/A	ophthalmic imaging platform system
Heidelberg Eye Explorer	Heidelberg Engineering, Germany	N/A	Version 1.9.13.0
78D standard ophthalmic non-contact slit lamp lens	Volk Optical Inc., Ohio, USA	V78C
Spectralis wide angle 55° lens	Heidelberg Engineering, Germany	50897-002
ultra widefield 102° lens	Heidelberg Engineering, Germany	50117-001
medetomidine hydrochloride 1 mg/mL (Domitor)	Provet AG, Lyssach, Switzerland	Swissmedic Nr. 50'590 - ATCvet: QN05CM91	anesthetic/analgesic
ketamine 50mg/ml (Ketalar)	Parke-Davis, Zurich, Switzerland	72276388	anesthetic
tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)	ISPI, Bern, Switzerland	N/A	pupil dilation
Omnican Insulin-50 0.5 ml G30 0.3 x 12mm	B. Braun Mesungen AG, Carl-Braun-Straße, Germany	9151125
hydroxypropylmethylcellulose (Methocel 2%)	OmniVision, Neuhausen, Switzerland	N/A
+4 dpt rigid gas permeable contact lens	Quantum I, Bausch + Lomb Inc., Rochester, NY	N/A	Base Curve: 7.20 to 8.40 mm Diameter: 9.00 / 9.60 / 10.20 mm Power: -25.00 to +25.00 Diopters
balanced salt solution (BSS)	Inselspital, Bern, Switzerland	N/A
silicon forceps	N/A	N/A
atipamezole 5 mg/mL (Antisedan)	Provet AG, Lyssach, Switzerland	N/A	α2 adrenergic receptor antagonist
GraphPad Prism 7	GraphPad Software, Inc, San Diego, CA, USA	N/A	statistical analysis software