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 without further intervention. SD-OCT provides information of retinal structure and retinal thickness by collecting backscattering data to create cross-sectional images of the retina, while SLO collects fluorescence data to produce stereoscopic high-contrast images of the retina. Nowadays, both techniques are increasingly used in experimental ophthalmology using small rodents^12,13,14,15 (or even zebrafish^16,17) and can provide both qualitative and quantitative information^{12,17,18,19,20,21}.

Accumulation of endogenous fluorophores like lipofuscins or the formation of drusen in the retina can be visualized by SLO as auto fluorescent signal. This feature makes SLO a valuable technique for diagnosis and monitoring of retinal diseases such as age-related macular degeneration or retinitis pigmentosa^22,23. In experimental ophthalmology, auto fluorescence imaging (AF) can be used for the detection of specific cell types in reporter mouse lines. For example, mice heterozygous for the expression of gfp under the promoter of Cx3cr1²⁴ are advantageous for in vivo visualization of microglial cells in the normal retina and for the investigation of microglia/macrophage dynamics in retinal disease²¹. Microglia are the resident macrophages of the retina, which play a crucial role on tissue homeostasis and tissue repair upon injury^1,25,26. Microglia activation in the retina has been reported in retinal injury, ischemia, and degeneration, suggesting a role of these cells in retinal disease^2,3,4,5,6.

The aim of the present protocol is to describe a relatively simple method for retinal imaging and measurement of retinal thickness using SD-OCT, and for visualization of gfp positive microglia cells in the Cx3cr1^gfp/gfp mouse retina using SLO (Heidelberg Spectralis HRA+OCT system). This protocol can be utilized for imaging and thickness measurements of healthy or diseased retinas in various mouse lines. Additionally, morphometric analyses can be performed for the identification and quantification of microglia numbers and microglia activation in the retina using SLO²¹. Microglia cells are associated with degenerative diseases in the central nervous system (CNS), including the retina^27,28,29. Thus, by combining the two methods used in the present protocol, correlation of microglia distribution and retinal degeneration can be made, which can facilitate monitoring disease severity or the effectiveness of therapeutic approaches in vivo.

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 inhalation.

NOTE: Perform each imaging session as quickly as possible (maximum 20 min), since cataract formation following anesthesia may hamper retinal visualization³⁰.

1. System Configuration

1. Turn on the computer.
2. Turn on the power supply. The message "Start acquisition module" will appear on the device`s control panel screen.
3. Double click on the software`s desktop shortcut to operate the software.
4. On the database view, click the "add patient" icon.
5. In the pop-up window, insert all necessary information (last name, first name, title, date of birth, sex, and patient-ID) and click "ok". On the pop window set the corneal curvature to 2 mm and click "ok".
6. Position a 78D standard ophthalmic non-contact slit lamp lens in front of the standard 30 ° optic and fasten it safely with tape.
7. Make sure that the filter lever on the left of the device is positioned on the indication "A" to allow both IR+OCT and AF imaging (Figure 1).

2. Mouse Preparation

1. Use a medetomidine hydrochloride dose of 0.75 mg/kg and a ketamine dose of 45 mg/kg in phosphate-buffered saline (PBS, pH 7.4) to prepare anesthesia solution. Thus, for a 20 g mouse, mix 15 µL of medetomidine hydrochloride with 18 µL of ketamine to a final volume of 50 µL in PBS. Store the solution at 4 °C for up to one week.
2. Grasp the mouse by the scruff and apply one drop of tropicamide 0.5% + phenylephrine hydrochloride 2.5% on each eye to achieve pupil dilation.
3. Keep the mouse restrained, and with an insulin syringe attached to a 30G needle, inject 50 µL of the anesthesia solution subcutaneously. Put the mouse back in its cage that is placed above a heating pad. Wait 3-5 min until the mouse is fully sedated. To assess anesthetic depth, pinch the tail of the mouse. Check the corneal reflex by gently touching the mouse cornea with a cotton swab. If no positive reflex is observed, continue with the imaging.
   NOTE: It is important to monitor respiratory rate during anesthesia. If the mouse is not fully sedated, the respiratory rate will increase upon a painful stimulus.
4. Hydrate the mouse cornea with an application of 2% hydroxypropylmethylcellulose drops on each eye.

3. SD-OCT

1. Place a warming patch of approximately 32 °C on the custom-made platform attached on the device`s chin rest (Figure 1).
2. To image the right eye, place the mouse on the left part of the platform (Figure 1). Ensure that the right orbit of the mouse faces the lens and its body lies prone on the left part of the platform.
3. Apply a drop of hydroxypropylmethylcellulose on the right eye and carefully position a +4 diopter rigid gas permeable contact lens on it (spherical power: -25.00 to +25.00 diopters). Store the contact lens in balanced salt solution (BSS). To grab the lens, use plastic or silicon forceps to avoid damage.
4. Press the yellow box on the right corner of the control panel display to start the acquisition module (green box on Figure 2A). The yellow box will turn green and the control panel menu will appear on the screen (Figure 2A).
5. For acquisition of B-scans, select the option IR+OCT on the control panel (Figure 2A). Selected settings are highlighted in blue on the control panel.
6. Select "Retina" under "Application and Structure" in the software and move the lens towards the mouse eye using the micromanipulator on the device (Figure 1). Before focusing onto the retina, check that the indication "OD" is selected on the left bottom part of the screen. The software automatically identifies the left (OS) and the right (OD) eye based on the position of the objective.
   NOTE: If the objective is positioned on the middle of the platform, an error message will appear on the bottom of the screen. In that case, re-position the mouse slightly until the software recognizes the correct eye.
7. With the focus knob, focus on the retina until the big vessels are clearly seen in the fundus image on the left of the computer screen. Move the camera by turning the micromanipulator left or right, to move the camera in that direction, or clockwise or counterclockwise, to move the camera up or down, respectively.
   NOTE: Reposition the mouse on the platform or move the lens upwards or downwards to achieve the preferred localization of the optic nerve head in the infrared image.
8. Turn the sensitivity knob counterclockwise to reduce or clockwise to increase the brightness of the fundus image. When optimal focus is achieved, an SD-OCT B-scan will appear on the right of the screen.
   NOTE: If the B-scan cannot be visualized by adjusting the focus, press the combination Ctrl+Alt+Shift+O on the keyboard. In the pop-up window, adjust the value of the reference arm until the B-scan appears on the screen.
9. On the bottom right of the screen, choose the single scan from the pattern menu (single line in Figure 2B).
10. Turn the micromanipulator of the device (Figure 1) left, right, forward, or backward (to turn the camera in that direction) to ensure that the B-scan is located between the top and bottom corners of the SD-OCT scan window.
11. Set the Automatic Real-Time (ART) value to at least 9 to obtain high quality images.
    NOTE: ART improves image quality by averaging several consecutive scans. The higher the "ART" value, the higher the signal-to-noise ratio and thus the image quality. However, by increasing the "ART" value, the acquiring time is also increased.
12. Press the "acquire" button (Figure 2) on the control panel screen and acquire the images.
13. Re-position the mouse to image the left eye and repeat steps 3.2-3.12.
14. Remove the contact lens with plastic/silicon forceps and place it into BSS.
15. Hydrate the mouse cornea with a fresh drop of hydroxypropylmethylcellulose and remove the excess with a tissue paper.
16. Remove the standard 30 ° optic by rotating it counterclockwise.
17. Mount the 55 ° lens and repeat steps 3.4-3.12 for each eye.

4. Auto Fluorescence Imaging

1. Without moving the mouse on the platform, select IR on the control panel.
2. With the focus knob, focus on the big retinal vessels.
3. On the control panel select AF.
4. Turn the sensitivity knob counterclockwise to reduce or clockwise to increase the brightness of the image.
5. Press the sensitivity knob and set the "ART" value to 67 or more to obtain high quality images.
6. When the set "ART" value has been reached, press "acquire" on the control panel to acquire the image. Press the sensitivity knob again to stop averaging. Adjust the focus, to visualize different retinal layers.
7. Remove the 55°  lens and mount the wide field 102°  lens. Follow steps 4.1–4.6 for both eyes of each mouse and for every mouse.
8. Click “Save images” on the left upper border of the window and then click “exit”. To save the images in the computer, select the mouse from the list on the right part of the screen by double clicking on the mouse name. Double click on each image separately and then click on the floppy disc icon. Save the image as .tif, .bmp, .jpg, or .png at the desired folder. To exit the software press “File” and “Exit”.

5. Anesthesia Reversal

1. Hydrate the mouse eyes with one drop of hydroxypropylmethylcellulose and return the mouse on a heating pad.
2. Prepare atipamezole solution in a dose of 2.25 mg/kg to reverse the sedative effects of medetomidine hydrochloride. For a 20 g mouse, add 9 µL of atipamezol in a final volume of 150 µL PBS. Store the solution at 4 °C for up to one week.
3. min after anesthesia injection (step 2.2), inject 150 µL of atipamezole solution subcutaneously.
4. Monitor the mouse until recovery from anesthesia. When the mouse is fully recovered (5-10 min after atipamezole injection) put it back in its cage. Recovery is indicated by the ability of the animal to turn its body when laid on one side, regaining of walking activity, and reacting in response to environmental stimulation.

6. Manual Retinal Thickness Measurement from SD-OCT Images

1. Open the OCT scan by double clicking on the name of the animal.
2. Open the B-scan obtained with the 30 ° or 55 ° lens.
3. Choose "thickness profile".
4. Press the "Edit Layer Segmentations" icon (Figure 2B). The software automatically identifies the inner limiting and base membrane.
5. If necessary, manually correct the position of the inner limiting and base membrane. To do so, choose the layer to be modified from the left side of the screen and then the red circle option (Figure 2B). Move the circle with the mouse button pressed to modify the line.
6. Modify the line to position the corresponding layer correctly. Click "Save and close" to exit the window.
   NOTE: Due to reflectivity of the choroid, the software may identify the base membrane incorrectly. Thus, it is preferable to define it manually before proceeding to retinal thickness measurements.
7. Choose "Retina" under the "Layer" option. A diagram of retinal thickness will appear on the bottom of the screen.
8. Click on a different position either on the diagram or on the B-scan to view the retinal thickness (indicated in µm) for the selected position.
9. Measure the retinal thickness in the desired distance from the optic nerve head and copy the values in a spreadsheet.
10. Repeat steps 5.1-5.9 for each mouse for both the 30 ° and the 55 ° lens.
11. Perform statistical analysis with desired software.

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 obser...

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

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