Name: Serie: Video-oculography in Mice
Uploaded: 2012-07-19T12:00:00.000+00:00
Duration: 9 min 43 s

We kindly thank the Netherlands Organisation for Health Research and Development (M.D.J, C.D.Z), The Netherlands Organisation for Scientific Research (C.D.Z), NeuroBasic (C.D.Z), Prinses Beatrix Fonds (C.D.Z), The SENSOPAC (C.D.Z), C7 (C.D.Z) and the CEREBNET (C.D.Z) program of the European Community for their financial support.

1. Preparation
The following experiments were conducted in accordance with The Duch Ethical Committee for Animal Experiments.
<ol>
	<li>Preparing mice for video-oculography. In order to measure eye movements of a mouse, the head of the mouse needs to be immobilized. Therefore, a pedestal construction is made on the skull of the mouse (Figure 1).
	<ol>
		<li>Anesthetize the mouse by a mixture of isoflurane (isofluran 1-1.5%; Rhodia Organique Fine LtD, France) and oxygen in a gas chamber. The excessive gas is scavenged. Maintain anesthesia via nose cone. Confirm depth of anesthesia via a toe pinch.</li>
		<li>Maintain the body temperature at 37 &deg;C with the use of an anal thermosensor and a heating pad (FHC, Bowdoinham, ME).</li>
		<li>Protect the eyes by covering them with an eye ointment (duratears, Alcon, Belgium). Shave the dorsal cranial fur, and clean the surgical area with a rotation of scrub and betadine or chlorhexidine solution.</li>
		<li>Make a middle line incision to expose the dorsal cranial surface of the skull. Make the surface clean and dry.</li>
		<li>Apply a drop of phosphoric acid (phosphoric acid gel etchant 37.5%; Kerr, CA) on the dorsal cranial surface of the skull from bregma to lambda. Remove the etchant after 15 seconds and make the cranial surface clean with saline and dry again.</li>
		<li>Apply on top of this etched cranial surface a drop of OptiBond prime (Kerr, CA) and air-dry it for 30 seconds.</li>
		<li>Place a drop of OptiBond adhesive (Kerr, CA) on top of the OptiBond prime and cure with light for 1 minute (Maxima 480 visible light curing unit; Henry Schein, USA).</li>
		<li>Cover the adhesive layer with a thin layer of Charisma composite (Heraeus Kulzer, Germany). Embed two connected nuts (diameter: 3 mm) in the composite. Cure the composite afterwards with light. When necessary, apply additional layers of composite and cure them with light.</li>
		<li>Administer buprenorphine (0.015 mg/kg, s.c) for post-operative analgesia. The animal should be back on its feet within approximately 5 min. Allow the mouse to recover in the home cage at room temperature for at least 3 days after the surgery. </li>
	</ol>
	</li>
	<li>Video-oculography setup for mice (Figure 2).
	<ol>
		<li>Place the mouse in the restrainer and fix his head to the restrainer by two screws (Figure 1). The mouse does not need to be anesthetized for this procedure. Restraining time should not exceed 1 hr/day.</li>
		<li>Mount the mouse head-and-body restrainer on an X-Y platform, which in turn is mounted upon the turntable (diameter: 60 cm). Using the X-Y platform the mouse head can be placed above the center of the turntable. The mouse can be moved over the pitch, yaw and roll axes. The head of the mouse is placed in the correct pitch, yaw and roll angle by aligning the eye using the visual image of the eye generated by the ISCAN system. Alternatively, the pedestal construction can be placed on the head of mouse in a stereotactic frame11.</li>
		<li>The turntable is attached to an AC servo-controlled motor (Harmonic drive AG, the Netherlands) and the position of the turntable is monitored by a potentiometer (Bourns inc., CA) attached to the turntable axis.</li>
		<li>A cylindrical surrounding screen (diameter: 63 cm; height: 35 cm) with a random dotted pattern (each element 2&deg;) covers the turntable; this drum is also equipped with an AC servo-controlled motor (Harmonic drive AG, the Netherlands). The position of the cylindrical screen is monitored by a potentiometer (Bourns inc., CA) attached to its axis and the screen can be lit by a halogen light (20 Watt). Both the surrounding screen and the turntable are driven independently.</li>
		<li>The movement of the turntable and surrounding screen is controlled by a computer that is connected to an I/O interface (CED limited, Cambridge, United Kingdom). Table and surrounding screen position signals are filtered (cut-off frequency: 20 Hz), digitized by the I/O interface and stored on this computer.</li>
		<li>The eye of the mouse is illuminated by three infrared emitters (600 mW, dispersion angle: 7&deg;, peak wavelength: 880 nm, RS components, the Netherlands). Two infrared emitters are fixed to the turntable and the third emitter is attached to the camera. This third emitter produces a reference corneal reflection (CR), which is used during the calibration procedure and during the eye movement recordings.</li>
		<li>An infrared CCD camera equipped with a zoom lens (Zoom 6000, Navitar inc., NY) is attached to the turntable and is focused on the mouse head in the center of the turntable. The camera can be unlocked and can be yawed about the turntable axis over exactly 20&deg; during the calibration procedure.</li>
		<li>The video signal is processed by an eye tracking system (ETL-200, ISCAN, Burlington, MA). The ISCAN system uses an algorithm to track the centers of the pupil and the reference CR. The system can track the pupil and reference CR in horizontal and vertical direction at a sample rate of 120 Hz.</li>
		<li>Reference CR position, pupil position and pupil size signals are digitized by the I/O interface and are stored in the same file as the table and surrounding screen position signals. The video pupil-tracking system induces a delay of the eye movement signals of approximately 27 ms.</li>
	</ol>
	</li>
</ol>
2. Calibrating and Measuring Eye Movements Using Video Pupil-tracking
The eye tracking system captures the movement of the pupil as a translational motion. The translational motion of the tracked pupil contains a translational component due to axial difference between the rotational center of the eye and the anatomical center of the eye (i.e. center of corneal curvature), and a rotational component due to the angular rotation of the eyeball. By subtracting the reference CR from the pupil movement/position, the undesired translational component is eliminated from the signal, resulting in a translational motion that is only due to the rotation of the eyeball. Although they are often very small, this subtraction also eliminates the translations between the head and the camera. The residual isolated translational motion is converted into the angular rotation of the eyeball by the following calibration method8,12. This calibration was performed prior to any eye movement experiment.
<ol>
	<li>Adjust the mouse head position to the camera in such a way that the video image of the pupil is situated at the middle of the monitor and that the representation of the reference CR is located on the vertical midline of the eye preferably direct above the pupil. Minimize the movements of the reference CR due to angular camera rotations, which can be accomplished by placing the center of the corneal curvature over the camera/table axis.</li>
	<li>Rotate the camera several times by +/- 10&deg; (i.e. 20 degrees peak to peak) around the vertical axis of the turntable. Use the positions of the tracked pupil (P) and the reference CR recorded in the extreme positions of the camera rotation to calculate the radius of rotation of the pupil (Rp; Rp = &Delta;/sin(20&deg;); where &Delta;=(CR-P), see Figure 3A).</li>
	<li>Due to the fact that the Rp value depends on the pupil size, a pupil size correction needs to be implemented12 (Figure 3B). Repeat step 2.2 many times under various illumination conditions (i.e. manipulating the pupil size; Figure 3C) in order to determine the pupil size - Rp relationship and compose an Rp correction curve (Figure 3D). The Rp value also depends on the vertical eye position. When the experiment will cause vertical eye movements then a correction of the calibration for vertical eye positions is highly recommendable13.</li>
	<li>Determine the angular position of the eye (E) by measuring the reference CR position, P position and the pupil size. The reference CR position is subtracted from the pupil position generating a translational free pupil position. By measuring the pupil size the Rp value can be extracted from the Rp correction curve and E can be calculated by using the following formula E= arcsin {(&Delta;1)/Rp} (Figure 4A; where &Delta;1=(P2-P1) and P1 and P2 are corrected by subtraction of the reference CR).</li>
	<li>A large repertoire of turntable and/or surrounding screen rotations can now be used to stimulate the oculomotor system. In order to perform video oculography in the dark, the mouse eye needs to be pretreated with a miotic drug to limit the pupil dilatation and allow pupil tracking under these circumstances. In our experiments, we use pilocarpine (4%, Laboratories Chauvin, France) to limit pupil dilatation in the dark.</li>
</ol>
3. Data Analysis
<ol>
	<li>Eye positions, table positions and surrounding screen positions are all converted into angular positions (see Figure 4B and formula in 2.4). Eye signals are corrected for their delay of 27 ms induced by the imaging processing of the pupil-tracking system.</li>
	<li>Angular positions of eye, table and surrounding screen are differentiated and filtered with a Butterworth low-pass filter using a cut off frequency of 20 Hz.</li>
	<li>Saccades are removed from the eye velocity signal using a detection threshold of 40&deg;/s. Data is removed starting from 20 ms before and up to 80 ms after crossing the detection threshold.</li>
	<li>Table, surrounding screen and eye velocity signals are averaged using each individual cycle in the trail (Figure 4C).</li>
	<li>Averaged signals are fitted with an appropriate function. In general, a sinusoidal velocity stimulation is used and the averaged cycles are fitted with sinus or cosinus function (Figure 4C). Then, the gain can be computed as the ratio of eye velocity to stimulus velocity, whereas the phase can be computed as the difference (in degrees) between the eye velocity and stimulus velocity.</li>
</ol>
4. Representative Results
Video-oculography can be used to investigate various forms of oculomotor performances (i.e. optokinetic reflex: OKR; vestibulo-ocular reflex: VOR; visually enhanced vestibulo-ocular reflex: VVOR) as well as motor learning (VOR adaptation; OKR adaptation). The OKR compensates for low-frequency disturbances using visual feedback. The OKR can be induced by rotating the well-illuminated surrounding screen (Movie 1). Rotating the surrounding screen over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg; shows how the optokinetic system is a more efficient compensatory mechanism in the low-frequency range than in the high-frequency range (Figure 5A). The VOR compensates for high-frequency head movements using signals from the vestibular organs. The VOR can be induced by rotating the animal (i.e. turntable) in the dark (Movie 2). Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg; demonstrates how the vestibulo-ocular system is more efficient in generating compensating eye movements in the high-frequency range than in the low-frequency range (Figure 5A). When the optokinetic and vestibulo-ocular system act in concert, images can be stabilized on the retina over a broad range of head movements. Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg;, while the surrounding screen is well-illuminated (Movie 3) shows how the eye generates "high gain" compensating movements over the entire frequency range (Figure 5A). All these gain and phase values are typical for mice, although gender14 and strain15,16,17 differences were reported.
The independent control over the turntable and the surrounding screen enables us to confront the mice with a mismatch between visual and vestibular information. After a long-term and uniform exposure of mismatched visual and vestibular information, the VOR of the mouse will change to compensate for the altered visual input (VOR adaptation; Movie 4). Rotating the turntable out of phase (i.e. 180&deg;) with the surrounding screen (1 Hz, 1.6&deg;) increases the VOR gain (Figure 5B). The maximal change in VOR gain, when using a one trial learning paradigm, is often reached after 30 minutes.
<img alt="Figure 1" src="/files/ftp_upload/3971/3971fig1.jpg" /> 
Figure 1. Schematic drawing of the mouse head-and-body restrainer. The body of the mouse is restrained using a plastic cylindrical tube with a diameter of 35 mm. The head of the mouse is immobilized by connecting the pedestal of the mouse to the iron bar with two screws. The iron bar makes an angle of 30 degree in order to position the head of the mouse in the normal pitch during ambulation. *, top view of the pedestal containing two nuts.
<img alt="Figure 2" src="/files/ftp_upload/3971/3971fig2.jpg" /> 
Figure 2. Schematic drawing of the mouse video-oculography setup.
<img alt="Figure 3" src="/files/ftp_upload/3971/3971fig3.jpg" /> 
Figure 3. Calibration of the video pupil-tracking system. A) The camera is rotated several times by +/- 10&deg; (i.e. 20 degrees peak to peak) around the vertical axis of the turntable. The tracked pupil (P) and the reference corneal reflection (CR) recorded in the extreme positions of the camera rotation are used to calculate the radius of rotation of the pupil (Rp). B) The radius of the pupil diameter is dependent on the size of the pupil. C) Example showing the effect of pupil size on pupil position during the calibration procedure (both measured in pixels (px)). D) Relationship between Rp and pupil diameter measured in a single mouse. The thirteen different pupil diameters were accomplished by altering the intensity of the surrounding light.
<img alt="Figure 4" src="/files/ftp_upload/3971/3971fig4.jpg" /> 
Figure 4. Measuring and analyzing eye movements using video pupil-tracking. A) The angular pupil position is calculated from radius of the pupil (Rp) and the position of the Pupil (P; corrected for CR position). B) Example of compensatory eye movement induced by stimulating the vestibular and visual system (visual enhanced VOR). The turntable was rotated sinusoidally at 0.6 Hz with an amplitude of 1.6&deg;, while the surrounding screen was well-illuminated. C) Analyses of the recording shown in B). Graph shows the averaged velocity trace of the turntable (blue) and pupil (red). These averaged traces were fitted with a sinusoidal function (black).
<img alt="Figure 5" src="/files/ftp_upload/3971/3971fig5.jpg" /> 
Figure 5. Performance and learning of the oculomotor system measured in one C57Bl6 mouse. A) Eye movements are generated by rotations of the surrounding screen (optokinetic reflex: OKR, top panels), by rotating the mouse in the dark (vestibulo-ocular reflex: VOR, middle panels) and by rotating the mouse in the light (visually-enhanced vestibulo-ocular reflex: VVOR, bottom panel) with frequencies ranging from 0.2 to 1.0 Hz at an amplitude of 1.6&deg;. The gain of the reflex was computed as the ratio of eye velocity to stimulus velocity (left panels) and phase of the reflex was computed from the phase difference between the eye velocity and stimulus velocity (right panels). B) Motor learning was accomplished by adaptively increasing the VOR using an out of phase training paradigm. The mouse was subject to a visuovestibular training paradigm in which the rotation of the mouse was out of phase (180&deg;) with the rotation of the surrounding screen (both rotating at 1.0 Hz, 1.6&deg;) for forty minutes. Every 10 minutes the VOR was tested (1.0 Hz, 1.6&deg;). In this mouse the out of phase training increased the VOR gain.
Movie 1. Animation showing the paradigm that induces OKR in mice <a href="https://www.jove.com/files/ftp_upload/3971/3971movie1.mov" target="_blank">Click here to view movie</a>.
Movie 2. Animation showing the paradigm that induces VOR in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie2.mov" target="_blank">Click here to view movie</a>.
Movie 3. Animation showing the paradigm that induces VVOR in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie3.mov" target="_blank">Click here to view movie</a>.
Movie 4. Animation showing the visuovestibular out of phase training paradigm that induces VOR adaptation (increase) in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie4.mov" target="_blank">Click here to view movie</a>.

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No conflicts of interest declared.

In order to obtain high-quality video eye movements recordings in mice several requirements are necessary. The calibration procedure needs to be performed in the above mentioned standardized matter. For example off-center calibration, when the pupil is not positioned on the vertical midline with the reference CR during the calibration procedure, will result in an underestimation of RP and consequently an overestimation of the eye movement. Furthermore, we recommend integrating the pupil size correction method in the calibration procedure12, because trials that show a very stable pupil size are very rare. Even a small stressor during the trial can already alter the pupil diameter substantially.
When designing an eye movement experiment, the following factors need to be taken into account or controlled for because they are known to affect the eye movement response: age13,18, gender14 and strain15,16,19. Furthermore, the experimental animal should have pigmented irises since pupil detection and tracking is impossible when the contrast between pupil and iris is too low, like in the BALB/c mouse. Extremely nervous or anxious animals need to be trained, prior to the experiment, to get used to the experimental set up and the restrained condition. This animal handling procedure results in less closure or semi-closure of the eyes and prevents the generation of eye fluids during the experiment, and consequently a better pupil tracking is accomplished.
Finally, acquiring and analyzing the data requires two to three hours per animal. Therefore, eye movement recordings will likely remain a specific procedure applied to selected mice and is not suitable as a high throughput screening test.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Isofluran</td>
 <td>Rhodia Organique Fine LTD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>FHC</td>
 <td>40-90-8</td>
 </tr>
 <tr>
 <td>Duratears</td>
 <td>Alcon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphoric acid gel</td>
 <td>Kerr</td>
 <td>31297</td>
 </tr>
 <tr>
 <td>Optibond prime</td>
 <td>Kerr</td>
 <td>35369</td>
 </tr>
 <tr>
 <td>Optibond adhesive</td>
 <td>Kerr</td>
 <td>35369</td>
 </tr>
 <tr>
 <td>Charisma composite</td>
 <td>Heraeus Kulzer</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Maxima 480 light curing unit</td>
 <td>Henry Schein</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AC servo-controlled motor</td>
 <td>Harmonic drive AG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cylindric screen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Halogen light (20 W)</td>
 <td>RS components</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potentiometers(precision)</td>
 <td>Bourns inc.</td>
 <td>6574</td>
 </tr>
 <tr>
 <td>Power 1401 (I/O interface)</td>
 <td>CED limited</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Computers</td>
 <td>Dell</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Infrared emmitters</td>
 <td>RS components</td>
 <td>195-451</td>
 </tr>
 <tr>
 <td>ETL-200</td>
 <td>ISCAN</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Zoom lens (zoom 6000)</td>
 <td>Navitar inc.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pilocarpinenitrate (minims)</td>
 <td>Laboratoire Chauvin</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

video-oculography in mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Watch this Scientific Journal Video about Video-oculography in Mice at JoVE.com

Video-oculography in Mice

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Research

JoVE Journal

Neuroscience

23.6K Aufrufe.  Erasmus MC, Rotterdam, The Netherlands. Video-EOG ist ein sehr quantitative Methode zur okularen motorischen Leistungsfähigkeit zu untersuchen sowie motorischen Lernens. Hier beschreiben wir, wie man Video-Okulographie bei Mäusen zu messen. Die Anwendung dieser Technik auf normalen, pharmakologisch behandelt oder gentechnisch veränderten Mäusen ist ein leistungsfähiges Werkzeug, um die Forschung zu Grunde liegenden Physiologie der motorischen Verhaltensweisen zu erforschen.

Serie: Video-oculography in Mice

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicine

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

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.

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.

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

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Evaluating Optokinetic Reflex Responses in Mice: A New High-Precision Protocol

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Author Spotlight: An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...