This work was supported by a grant of the Swiss National Science Foundation (SNSF; #320030_156019). The authors received nonfinancial support from Heidelberg Engineering GmBH, Germany.

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 ...

<ol>
	<li>Madeira, M. H., Boia, R., Santos, P. F., Ambrosio, A. F., Santiago, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+microglia-mediated+neuroinflammation+to+retinal+degenerative+diseases.">Contribution of microglia-mediated neuroinflammation to retinal degenerative diseases.</a> Mediators Inflamm. , 673090 (2015).</li><li>Ng, T. F., Streilein, J. 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I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia-mediated+IGF-I+neuroprotection+in+the+rd10+mouse+model+of+retinitis+pigmentosa.">Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmentosa.</a> Invest Ophthalmol Vis Sci. 52 (12), 9124-9130 (2011).</li><li>Zhang, C., Lam, T. T., Tso, M. 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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 time10,14,17,18,21. With this protocol, Cx3cr1...

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 rodents12,13,14,15 (or even zebrafish16,17) and can provide both qualitative and quantitative information12,17,18,19,20,21.
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 pigmentosa22,23. 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 injury1,25,26. Microglia activation in the retina has been reported in retinal injury, ischemia, and degeneration, suggesting a role of these cells in retinal disease2,3,4,5,6.
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 Cx3cr1gfp/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 retina27,28,29. 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.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

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-Cx3cr1gfp/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 diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/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 &#181;m and 6 &#181;m, 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.

Using the protocol presented here, SD-OCT scans and SLO images were obtained from Cx3cr1gfp/gfp mice in the same imaging session. Figure 3 includes representative SD-OCT single scans obtained with a 30 &#176; or a 55 &#176; lens (Figure 3A) and representative SLO images obtained with a 55 &#176; or a 102 &#176; lens, where gfp positive microglia cells are visualized. Higher reflectivity of the choroid is obser...

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

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.

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

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

The overall goals of this procedure are to use spectral-domain optical coherence tomography and scanning laser ophthalmoscopy to obtain information on retinal thickness and microglial cell distribution, respectively. This method can help answer key questions in the experimental ophthalmology field about the correlation between retinal abnormalities and microglial cell accumulation. The main advantages of this technique are that this information can be obtained in real-time in a non-invasive manner.After confirming a lack of response to corneal swab, place the mouse in the prone position on the left side of the platform, with the right orbit facing the lens. Apply a drop of hydroxypropyl methylcellulose in a plus four diopter, rigid, gas-permeable contact lens onto the right eye and start the acquisition module. For B scans, select the Infrared plus Optical Coherence Tomography option Under Application and Structure, select Retina and use the micro-manipulator to move the lens towards the mouse eye.Before focusing on the retina, confirm that the oculus dexter indicator is selected, then use the focus knob to zoom into the retina until the big vessels are clearly visible in the fundus image on the left side of the monitor screen. Use the micro-manipulator to adjust the position of the camera, as necessary, turning the sensitivity knob to reduce or increase the brightness of the fundus image, as appropriate. Select the Line Scan from the Pattern menu and use the micro-manipulator to move the B scan between the top and bottom corners of the spectral-domain optical coherence tomography, or SD-OCT, scan window.Set the Automatic Real Time value to at least nine to obtain a high image quality and click Acquire. When all of the images have been obtained, transfer the contact lens from the eye into fresh-balanced salt solution and hydrate the cornea with a fresh drop of hydroxypropyl methylcellulose. After the left eye has been imaged, rotate the standard 30 degree optic in the counter-clockwise direction to remove it and mount the 55 degree lens for a second round of imaging.To assess the auto fluorescence, without moving the mouse, select Infrared on the control panel and focus on the big retinal vessels. Select auto fluorescence imaging and use the sensitivity knob to adjust the image brightness, as necessary. Press the sensitivity knob once and set the automatic real-time value to at least 67.When the automatic real-time value has been reached, acquire the image and press the sensitivity knob a second time to stop the averaging. Adjust the focus to visualize the different retinal layers, as experimentally appropriate. When all of the images have been obtained, on to wide-field 102 degree lens onto the optic and image the auto fluorescence in each eye, as just demonstrated.To measure the manual retinal thickness in each image, double-click on the name in the first experimental animal to open the OCT scan. Open a B scan obtained with the 30 or 55 degree lens and select the thickness profile. Click the Edit Layer Segmentations icon.The software will automatically identify the inner limiting and base membranes. To manually correct the position of the membranes, select the layer to be modified, as well as the red circle option. Holding down the mouse button, move the circle to modify the line until the corresponding layer is correctly positioned and click Save and Close to exit the window.Under the Layer option, select Retina and click on a different position on the diagram to view the retinal thickness for the selected position. Then measure the retinal thickness and the desired distance from the optic nerve head and export the values into a spreadsheet. In these representative SD-OCT single scans from a mouse homozygous for the expression of gfp under the Cx3cr1 promoter, the retinal architecture is clearly visualized in both the 30 degree and 55 degree lens images.However, a high reflectivity of the choroid is observed in the scans obtained with the 30 degree lens. Following SD-OCT, scanning laser ophthalmoscopy allows the visualization of the individual gfp positive microglial cells in the retina using a 55 degree or a 102 degree lens with larger fundus area coverage obtained with the 102 degree lens. In the SD-OCT scans, after manual correction of the inner limiting and base membrane retinal boundaries, a good correlation of the retinal thickness measurement is typically observed between the 30 and 55 degree lenses when the same distance from the optic nerve head is measured.Once mastered, this technique can be completed in less than fifty minutes if it's performed properly. After its development, this technique paved the way for researchers in the experimental ophthalmology field to explore retinal pathology in small rodents in vivo.

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10.5K Views.  Bern University Hospital and University of Bern. The overall goals of this procedure are to use spectral-domain optical coherence tomography and scanning laser ophthalmoscopy to obtain information on retinal thickness and microglial cell distribution, respectively. This method can help answer key questions in the experimental ophthalmology field about the correlation between retinal abnormalities and microglial cell accumulation. The main advantages of this technique are that this information can be obtained in real-time in a non-invasive m...