This work was supported by grants of the Dr. Robert Pfleger-Foundation and the Ilselore Luckow-Foundation, as well as Biogen and Novartis to PA. Figure 1B was reproduced from &#34;Whole-body positional manipulators for ocular imaging of anaesthetized mice and rats: A do-it-yourself guide. Dietrich, M., Cruz-Herranz, A., Yiu, H., Aktas, O., Brandt, A. U., Hartung, HP., Green, A., Albrecht, P. BMJ Open Ophthalmology. 1 (1), e000008, 2017&#34; with permission from BMJ Publishing Group Ltd.

All animal procedures were performed in compliance with the experimental guidelines approved by the regional authorities (State Agency for Nature, Environment and Consumer Protection; reference number 84-02.04.2014.A059) and conform to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the European directive 2010/63/EU on the protection of animals used for scientific purposes.
1. Confocal Scanning Laser Ophthalmoscopy-optical Coherence Tomography
NOTE: The protocol for cSLO-OCT measurement is adaptable for all strains of laboratory mice and rats.
<ol>
	<li>Set-up and pre-imaging preparations 
	NOTE: The system configuration of the OCT device used in this protocol has already been described elsewhere31.</li>
	<li>Rodent preparation for inhalant anesthesia
	<ol>
		<li>Place the rodent in an induction chamber and set the vaporizer to an isoflurane concentration of 2% at 2 L/min O2.</li>
		<li>Check if the rodent is anesthetized by pinching the tail, remove it from the chamber and wrap it in paper towel to keep it warm.</li>
		<li>Place the rodent in the custom holder33 and hook the maxillary incisors on the integrated bite bar of the mouth piece, connected to the vaporizer (2.5% isoflurane at 2 L/min O2).</li>
		<li>Apply one drop of Phenylephrine 2.5%-Tropicamide 0.5% on each eye for pupillary dilation.</li>
		<li>Wipe off any excess liquid of eye drops after 1 min and lubricate the eyes with methyl-cellulose based ophthalmic gel (e.g., hypromellose 0.3% eye drops) to avoid drying out and turbidity of the cornea.</li>
		<li>Place custom contact lens (+4 diopters) on the mouse eye by hand or using forceps. Cover the rat eye with a glass plate (e.g., round 12 mm diameter glass coverslip) without optical properties to assure a plane surface. 
		NOTE:&#160;Monitor respiratory rate during anesthesia. Increase or decrease Isoflurane concentration if required.</li>
	</ol>
	</li>
	<li>Measurement and analysis 
	NOTE: Make sure to perform and report the OCT measurements in line with the APOSTEL recommendations34 and perform quality control according to the OSCAR-IB consensus criteria35. As these recommendations have been developed for human OCT images, some criteria are not or only partially applicable.
	<ol>
		<li>To image the left eye, position the holder as presented in Figure 1A to ensure that the left eye bulb of the rodent faces the camera.</li>
		<li>Press the Start button in the right corner of the control panel display to start the acquisition mode.</li>
		<li>Set the filter lever to R and select BR+OCT for Blue reflectance fundus imaging and B-scan acquisition on the control panel.</li>
		<li>Set the focus distance to approx. 38 diopters using the focus knob on the back of the camera and zoom in on the retina until the OCT scan is visible on the screen. 
		NOTE: At the first measurement, the reference arm has to be adapted for rodent measurement. Press the combination Ctrl+Alt+Shift+O and adjust the value of the reference arm in the open window until the OCT-scan appears on the screen.</li>
		<li>To ensure a beam path through the middle of the pupil with an orthogonal angle to the retina in all planes, position the optic disc in the middle of the illuminated field (BR) and adjust the horizontal and vertical line B-scans to a horizontal level by rotating/turning the holder (Figure 1B) or moving the camera.</li>
		<li>Select the volume scan mode and set it to 25 B-scans in high-resolution mode at 50 automatic real-time tracking (ART, rasterized from 50 averaged A-Scans) on the software screen.</li>
		<li>Center the middle of the volume scan grid on the optic disc and start acquisition by pressing the black sensitivity knob and then AQUIRE on the control panel.</li>
		<li>Set the filter lever to A, select Blue Auto Florescence (BAF) on the control panel and adjust image brightness with the sensitivity knob. Press the sensitivity knob and then AQUIRE to image fluorescent cells (e.g., EGFP) or auto fluorescent deposits.</li>
		<li>Apply ophthalmic gel on the eye of the rodent to prevent dehydration and put the animal in a separate cage with a heat source.</li>
		<li>Supervise the rodent until it is fully recovered from anesthesia, in a separate cage and individually housed. When the animal is ambulatory, return it to the home cage.</li>
		<li>For analysis of the volume scans, use the automated segmentation of the OCT device&#8217;s software by right-clicking on the scan and select Segmentation then All Layers. Make sure that the quality of the OCT images is sufficient and define quality cutoffs for each set of experiments, e.g., &#62;20 decibels.</li>
		<li>Perform manual correction of the layers by double clicking on the desired scan, select Thickness Profile and click on Edit Layer Segmentations. Select one layer, e.g., press ILM for Inner limiting membrane, and, if necessary, correct the green line by moving the red dots by drag and drop to the correct position. 
		NOTE: Make sure the investigator performing the manual correction is blinded for the experimental groups.</li>
		<li>Select the tab Thickness Map and choose the 1, 2, 3 mm early treatment of diabetic retinopathy study (ETDRS) grid. Center the inner circle on the optic disc (Figure 2, left).</li>
		<li>Calculate the thickness of retinal layers from the thickness values provided by the software for the different retinal sectors of interest. To compute the mean thickness values from volume scans, use the whole 1, 2, 3 mm ETDRS grid, which covers an angle of approximately 25&#176;, excluding the inner 1 mm circle, which contains the optic disc (Figure 2, right).</li>
		<li>Perform the statistical analysis using adequate software. If both eyes of an animal are included, consider a statistical model accounting for within subject inter-eye correlations (e.g., generalized estimating equations or mixed linear models), as the eyes of one subject are statistically dependent36.</li>
	</ol>
	</li>
</ol>
2. Optokinetic Response
NOTE: In the following, a detailed manual for OKR measurements of mice and rats is provided, which can be adapted to individual specific needs.
<ol>
	<li>Set-up and pre-measurement preparations
	<ol>
		<li>Turn on the computer. After the system has booted, turn on the screens of the testing chamber as described in more detail elsewhere37.</li>
		<li>Select a suitable platform for the measurement of mice or rats. 
		NOTE: The platform size is selected based on the body size of the rodent. The animal should be able to sit properly on the platform without the ability to walk around.</li>
		<li>Open the pre-settings window by double-clicking on the software, select New group and choose the group name, the number of subjects, the species and strains. Select a variable stimulus: spatial/temporal frequency, contrast sensitivity, speed or orientation in the drop-down menu, then press Create New Group.</li>
		<li>Focus on the platform by manipulating the focus ring of the camera on top of the chamber and calibrate the system by aligning (drag and drop) the red circle around the black circle on the platform.</li>
	</ol>
	</li>
	<li>Measurement and analysis
	<ol>
		<li>Place the animal on the platform, let it adapt to the environment for ~5 min. Lift the animal back on platform if it drops (Figure 3A).</li>
		<li>Select Subject number and Condition on the top right corner of the software screen (Figure 3B). One stimulus is variable, the other stimuli are kept constant. This is confirmed by the Open lock or Closed lock symbol next to the stimulus.</li>
		<li>Start measurement by selecting &#9668; for Yes or &#9632; for No, if the animal tracks or does not track, respectively. 
		NOTE: Clockwise tracking corresponds to the left and counterclockwise tracking to the right eye. The software randomly changes the direction of the moving grid.</li>
		<li>Select the step size of the stimulus manually by clicking on the Up and Down arrows next to the variable stimulus or let it adapt automatically by the software if stimulus threshold converges.</li>
		<li>For optimal results, animate the animal, e.g., by high whistling sounds and blanking, by clicking the black or white box symbol on the software screen. Perform these actions repeatedly in the case of prolonged measurements.</li>
		<li>For data analysis, select the Summary tab and click on File | Export Table/Graph to export the desired data set.</li>
		<li>Perform the statistical analysis using the desired software (see also Step 1.3.15).</li>
	</ol>
	</li>
</ol>

Unrelated to the work presented the authors declare the following financial disclosures: 
Michael Dietrich received speaker honoraria from Novartis. Andr&#233;s Cruz-Herranz is a postdoctoral fellow of the National Multiple Sclerosis Society. Ari J. Green served on the scientific advisory board of MedImmune, Novartis, OCTIMS, Inception 5 Biosciences, and Bionure; is an associate editor of JAMA Neurology; was an editorial board member of Neurology; holds a patent for remyelination molecules and pathways; consulted for Inception 5 Sciences; received research support from Novartis Pharma OCTIMs, Inception Sciences SRA, NINDS, NIA, National MS Society, Sherak Foundation, and Hilton Foundation; holds stock or stock options in Inception 5; and served as an expert witness at Mylan v Teva Pharma. Hans-Peter Hartung has received fees for serving on steering committees from Biogen Idec, GeNeuro, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, MedImmune, Bayer HealthCare, Forward Pharma, and Roche, fees for serving on advisory boards from Biogen Idec, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, and Roche, and lecture fees from Biogen Idec, Sanofi Genzyme, Merck, Novartis Pharmaceuticals, Octapharma, Opexa Therapeutics, Teva Pharmaceuticals, MedImmune, and Roche. Philipp Albrecht received compensation for serving on Scientific Advisory Boards for Ipsen, Novartis, Biogen; he received speaker honoraria and travel support from Novartis, Teva, Biogen, Merz Pharmaceuticals, Ipsen, Allergan, Bayer Healthcare, Esai, UCB and Glaxo Smith Kline; he received research support from Novartis, Biogen, Teva, Merz Pharmaceuticals, Ipsen, and Roche. The other authors report no disclosures.

This protocol provides an instruction for the thickness measurements and the examination of visual function in rodents. Visual readouts are increasingly used in translational research18,26,38,39,40 and are easily transferable to clinical trials. The significant advantage of OCT in comparison to histological investigations in animal experiments is that longitudinal analyses are possible allowing the investigation of dynamic pathological processes, largely reducing the variability and the number of animals needed per study. Furthermore, in vivo imaging with OCT is not subject to fixation, cutting or staining artifacts, which may affect the layer thickness in histological investigations.
However, the orthogonal orientation of the laser beam in all planes in relation to the retina is a critical step to ensure the quality and reproducibility of the thickness values. It requires some training of the investigator and is mandatory before the acquisition of OCT scans. Additionally, as the commercial devices are built for human applications, the quality of rodent OCT images is still inferior compared to B-scans of human patients. In the authors&#39; experience, it may be difficult to distinguish the different inner retinal layers (retinal nerve fiber layer, ganglion cell layer and inner plexiform layer) during manual correction. We therefore recommend analyzing these layers as a compound readout (IRL).
The experimental setup provides an option for volatile anesthesia, e.g., inhalant isoflurane, which is, in our experience, safer and easier to control than injectable anesthesia, e.g., ketamine-xylazine41,42 and reduces the risk of premature awakening of rodents in case of longer acquisition times (e.g., when performing imaging of fluorescently labelled cells). In a preliminary study, volume scans were identified as the protocols with the highest validity and reliability. The inter-rater and test retest reliability was excellent when volume scans excluding the central part containing the optic disc were assessed with ICC (intra-class correlation coefficient) values above 0.85 for all assessments.
The measurement of the optokinetic response is based on the involuntary optokinetic reflex, which occurs in response to a continuously moving field. In rodents, in contrast to other species, the movement involves not only the eyes, but the whole head, which can easily be detected using the camera.
Distinguishing between &#34;tracking&#34; or normal behavioral movements of the animals requires some training of the investigator and it is important to be blinded for the experimental group. In addition, the animals need an adaption phase to accommodate to the experimental setting and during long-time measurement protocols, the animals have to be animated repeatedly to assure that &#34;no tracking&#34; is due to reaching the OKR threshold and not to decreasing attention. There is also a significant strain variability regarding the visual function of laboratory mice and rats43,44. The visual acuity of the rodent should therefore be evaluated before they are tested and some strains, such as SJL mice, may not even be suitable for OKR measurements, as they are homozygous for the allele Pde6brd1 (retinal degeneration 1).
In summary, the examination of retinal morphology and visual function in animal models allows for non-invasive, longitudinal investigations of structural and functional damage occurring in the context of EAE and may be helpful in other models involving the visual system, including but not limited to the models of retinopathies or optic nerve injury.

The examination of the visual pathway, as a part of the central nervous system, has been proven to be an effective starting point in addressing not only ophthalmologic1,2,3,4,5, but also neurologic6,7,8,9,10,11,12,13,14,15,16 questions. In recent years, OCT and OKR have been identified as useful analytic, non-invasive tools to evaluate a large variety of retinopathies and retinal manifestations in various rodent models17,18,19,20,21,22,23,24,25. OCT allows for fast and high resolution in vivo visualization of the retinal morphology and structure in mice and rats, with results in good accordance with histological sections of the animals retinae26. OKR constitutes a fast and robust method to quantitatively assess visual function.
Many OCT devices allow simultaneous confocal scanning laser ophthalmoscopy (cSLO) imaging with different wavelengths, which provides diagnostic information about retinal pathologies, i.e., visualization of lipofuscin deposits or alterations of the retinal pigment epithelium27. Furthermore, in vivo imaging of fluorescence labelled cells in transgenic animals is possible28,29,30,31,32. However, the application of OCT technology in rodent models is still challenging, mainly because of the small eye size. Several commercially available devices require adaptations and often a different size of holder is required to image the animals of different species. Additionally, animals require anesthesia for measurement.
OKR devices can be used to assess the visual function in rodents. The animals are placed on a platform in the center of an actual or virtual cylinder displaying a moving grating, which the animals track with reflexive head and neck movements. This optokinetic response is reduced or eliminated in the case of the reduction or loss of visual function.
The aim of this protocol is to present a manual for the measurement of retinal thickness using a commercially available OCT device with a custom holder providing inhalant anesthesia. The protocol illustrates how to analyze volume scans using the software provided by the manufacturer. For visual testing, the aim is to provide instructions on how to use a commercially available system to assess the OKR.

<table><tbody><tr><td>Heidelberg Spectralis HRA+OCT system&amp;nbsp;</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 1.9.10.0</td></tr><tr><td>blue 25D non-contact&amp;nbsp; lens</td><td>Heidelberg Engineering, Germany</td><td>N/A</td><td>lens for rodent mesurement</td></tr><tr><td>OptoMotry</td><td>CerebralMechanics Inc., Canada</td><td>N/A</td><td>system for visual function analysis</td></tr><tr><td>OptoMorty HD software</td><td>CerebralMechanics Inc., Canada</td><td>N/A</td><td>Version 2.1.0</td></tr><tr><td>Inhalation Anesthetic Isoflurane</td><td>Piramal Critical Care, Bethlehem, PA, USA&amp;nbsp;</td><td>803250</td><td>inhalation anesthetic</td></tr><tr><td>Phenylephrin 2.5%-Tropicamide 0.5%&amp;nbsp;</td><td>University Hospital D&amp;uuml;sseldorf, Germany</td><td>N/A</td><td>pupillary dilation&amp;nbsp;</td></tr><tr><td>Visc-Ophtal</td><td>Dr. Robert Winzer Pharma GmbH, Berlin, Germany</td><td>58407</td><td>ophthalmologic eye gel</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software Inc, San Diego, CA, USA</td><td>N/A</td><td>statistical analysis software, Version 5.00</td></tr><tr><td>IBM SPSS Statistics</td><td>IBM Corporation, Armonk, New York, USA</td><td>N/A</td><td>statistical analysis software, Version 20</td></tr></tbody></table>

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.

Using 3rd generation OCT imaging in myelin oligodendrocyte glycoprotein (MOG) peptide induced experimental autoimmune encephalomyelitis (EAE) mouse models, high-resolution morphological sections of the mouse retina were obtained. Using this technology, the protective capacities of different substances were demonstrated17. The thickness values of the inner retinal layers (IRL) obtained are in good accordance with the numbers of retinal ganglion cells (RGC) obtained by histological staining of retinal wholemounts (Figure 4).
OKR monitoring provides a functional readout of the neurodegeneration seen by OCT. In these experiments, visual function assessed as spatial frequency by OKR, and neuroaxonal damage assessed as IRL thinning by OCT, were in close correlation17. Various protocols can be employed to examine the visual acuity by changing the spatial or temporal frequency, contrast sensitivity, orientation or speed of the moving grid. In the EAE model, an improved spatial frequency of 0.05 cycles/degree (c/d) of animals treated with substance 1 was detected compared to untreated MOG-EAE mice (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58571/58571fig1.jpg" /> 
Figure 1: Custom holder for OCT measurement. (A) OCT imaging of a C57BL/6J mouse using the custom holder33 and (B) rotational axis around the rodent eye. Rotation in the transverse plane (left) and in the axial plane (right) is demonstrated. This figure has been modified from Dietrich, M. et al.33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58571/58571fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58571/58571fig2.jpg" /> 
Figure 2: OCT post acquisition analysis. &#34;1, 2, 3 mm&#34; ETDRS grid on the 25 B-scan volume protocol (left). The thickness of retinal layers is provided for the different retinal sectors by the software (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58571/58571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58571/58571fig3.jpg" /> 
Figure 3: OKR measurement of mice and stimulus settings. (A) Top view through the camera analyzing a C57BL/6J mouse on the platform in the chamber. (B) User interface and settings of the OKR software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58571/58571fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58571/58571fig4.jpg" /> 
Figure 4: C57BL/6J mice with MOG EAE show an attenuated disease course when treated with substance 1 compared to untreated controls. (A) The degeneration of the inner retinal layers is reduced (B) and the clinical EAE score is attenuated during the EAE course when substance 1 was administered. Mice were scored daily, and OCT measurements were performed monthly over a period of 120 days. The graphs represent the mean and standard error of at least ten animals per group. (*p&#160;&#60; 0.05, ***p&#160;&#60; 0.001, area under the curve compared by ANOVA with Dunnett&#39;s post hoc test). (C) The IRL thickness change is in good accordance with RGC loss (***p&#160;&#60; 0.001, by ANOVA with Dunnett&#39;s post hoc test compared to MOG untreated mice). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58571/58571fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58571/58571fig5.jpg" /> 
Figure 5: OKR measurement of C57BL/6J mice with MOG-EAE. (A) OKR reveals an improved visual acuity of animals treated with substance 1 compared to untreated MOG EAE mice measured by spatial frequency threshold testing over a period of 120 days. The graphs represent the mean and standard error of at least six animals per group (**p&#160;&#60; 0.01, ***p&#160;&#60; 0.001, area under the curve compared by ANOVA with Dunnett&#39;s post hoc test). (B) Image of a C57BL/6J mouse in the testing chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58571/58571fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats at JoVE.com

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

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 ...

using-optical-coherence-tomography-optokinetic-response-as-structural

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.9K Views.  Heinrich-Heine-University Düsseldorf. Deze methoden kunnen helpen bij het beantwoorden van belangrijke vragen op het gebied van neurologie en oogheelkunde, en helpen bij het evalueren van een grote verscheidenheid aan retinopathieën en netvliesmanifestaties in verschillende knaagdiermodellen. Het belangrijkste voordeel van deze technieken is dat niet-invasieve longitudinale onderzoeken kunnen worden uitgevoerd, waardoor de variabiliteit wordt verminderd en het aantal dieren dat nodig is voor de experimenten wordt verminderd. Dez...

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

Using Looming Visual Stimuli to Evaluate Mouse Vision

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina through the lateral geniculate nucleus to the visual cortex, the system is complex. Cellular and molecular studies have been conducted to elucidate the mechanisms underpinning visual processing and, by extension, disease mechanisms. These studies may contribute to the development of artificial visual systems. To validate the results of these studies, behavioral vision testing is necessary. Here, we show that the looming stimulation experiment is a reliable mouse vision test that requires a relatively simple setup. The looming experiment was conducted in a large enclosure with a shelter in one corner and a computer monitor located on the ceiling. A CCD camera positioned next to the computer monitor served to observe mouse behavior. A mouse was placed in the enclosure for 10 minutes and allowed to acclimate to and explore the surroundings. Then, the monitor projected a program-derived looming stimulus 10 times. The mouse responded to the stimuli either by freezing or by fleeing to the hiding place. The mouse&#39;s behavior before and after the looming stimuli was recorded, and the video was analyzed using motion tracking software. The velocity of the mouse movement significantly changed after the looming stimuli. In contrast, no reaction was observed in blind mice. Our results demonstrate that the simple looming experiment is a reliable test of mouse vision.

To examine mouse vision, we conducted a looming test. Mice were placed in a large square arena with a monitor on its ceiling. The looming visual stimulus consistently evoked freezing or flight reactions in the mice.

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina ...

Behavior

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...

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 ...

Medicine

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

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 ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

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

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