Project Source: Natural Science Foundation of Guangdong Province (2018A0303130306). The authors would like to thank the Ophthalmic Research Laboratory, Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong for funding and materials.

The operations were performed following the Statement on the Use of Animals in Ophthalmic and Vision research from the Association for Research in Vision and Ophthalmology. The experimental design was approved by the institutional animal Ethics Committee (Medical Ethics Committee of JSIEC, EC 20171213(4)-P01). Two-month-old C57BL/6J mice and Vldlr knockout mice were used in this study. There were 7 mice in each group, all of which were female and weighed 20 g to 24 g.
1. Experimental conditions
<ol>
	<li>Assign the mice to two groups: an experimental group consisting of Vldlr knockout mice and a control group consisting of C57BL/6J mice.</li>
	<li>Feed the mice with food and water conventionally.</li>
	<li>Raise the mice in the animal laboratory under stable conditions of room temperature (22 &#176;C), humidity (50-60%), light-dark cycle (12 h-12 h), and room light intensity (350-400 lux).</li>
	<li>Prepare the experimental equipment: optical coherence tomography with confocal scanning laser ophthalmoscope (cSLO) for small animals (Figure 1A).</li>
	<li>Prepare all materials required for the experiment (Figure 1B) and weigh the mice (Figure 1C).</li>
</ol>
2. Information records
<ol>
	<li>Record the information: group, code, date of birth, age, sex, weight, and anesthetic dosage.</li>
</ol>
3. Instrument startup and testing
<ol>
	<li>Switch on the computer and start up the software.</li>
	<li>Click the Test program button to complete the test program.</li>
	<li>Turn on the thermostat and preheat it to the temperature of 37 &#176;C.</li>
	<li>Start the OCT module procedure after the program testing.</li>
	<li>Create a new subject and fill in the mouse information.</li>
	<li>Preheat the electric blanket and cover it with surgical towels.</li>
</ol>
4. Anesthesia
<ol>
	<li>Use lyophilized anesthetic powder containing Tiletamine and Zolazepam to prepare the anesthetic mixture. 
	NOTE: Follow local animal ethics committee recommendations for the choice, dosage, and route of anesthesia administration. Anesthetize the animal with an anesthetic that will provide immobility and loss of pain perception for at least 1 hour, after which the animal recovers quickly. Dosage should be based on the length of experiment time, animal weight, and other factors.</li>
	<li>Anesthetize the animal using the prepared anesthetic mixture. Ensure to keep the animal warm during the entire procedure until recovery.</li>
</ol>
5. Application of mydriatic drops
<ol>
	<li>Achieve manual restraint of the mouse by the scruff, make the eyeball protrude slightly, and rotate the mouse head with one eye facing upward.</li>
	<li>Apply the mydriatic drops to dilate the pupils (Figure 2A).</li>
	<li>Check for pupil dilation after 10 min.</li>
</ol>
6. Placement of the mouse
<ol>
	<li>Place a mouse on an electric blanket platform.</li>
	<li>Coat both eyes with medical sodium hyaluronate gel (Figure 2B).</li>
	<li>Screw a 60 D double spherical lens (preset lens) on the cSLO device (Figure 1A-5, 6).</li>
	<li>Place a 100 D contact lens on the mouse cornea with the concave side touching the sodium hyaluronate gel on the corneal surface (Figure 2C, D and Figure 3A-II).</li>
	<li>Place the mouse on the small, constant-temperature animal platform and keep the eye 1-2 mm away from the lens of the cSLO device (Figure 3A).</li>
	<li>Adjust the angle of the contact lens with forceps to keep the pupil in the center of the lens.</li>
	<li>Fine-tune the adjustments to the head to make the eye face straight ahead.</li>
</ol>
7. Confocal Scanning Laser Ophthalmoscope (cSLO)
<ol>
	<li>Click the OCT button, choose the mouse module, and start the cSLO program (Figure 4B).</li>
	<li>Select the IR mode (light source: red light), and adjust the parameter (range: 2047, Figure 4D).</li>
	<li>Select the eye to be examined (right eye: Figure 4C-1; left eye: Figure 4C-2).</li>
	<li>Control the lever and move the preset lens towards the contact lens slowly.</li>
	<li>Adjust the diopter value until the posterior pole imaging is clear (Figure 4E).</li>
	<li>Make further adjustments to align the image of the retinal posterior pole, centering it at the optic nerve head.</li>
</ol>
8. Optical coherence tomography (OCT)
<ol>
	<li>Start the OCT program (Figure 4G).</li>
	<li>Click the progress bar up and down until the OCT image appears (Figure 4H).</li>
	<li>Adjust parameters: Range Min (Figure 4I) = 0-20, Range Max (Figure 4J) = 40-60.</li>
	<li>Adjust the preset lens distance and position direction until an ideal OCT image is obtained.</li>
	<li>Select the scanning position by moving the standard line in the cSLO (Figure 4M).</li>
	<li>Start scanning from the optic nerve head.</li>
	<li>Collect images in the same order for each eye: horizontal line: optic nerve head &#8594; superior &#8594; inferior; vertical line: optic nerve head &#8594; nasal &#8594; temporal.</li>
	<li>Collect images from four directions.</li>
	<li>Click Average to overlay the cSLO and OCT image signals (Figure 4F and Figure 4O).</li>
	<li>Click the shot button to acquire the SLO-OCT image (Figure 4P).</li>
	<li>Save and export all the images (Figure 4Q, R).</li>
</ol>
9. The end of the experiment (after the OCT examination)
<ol>
	<li>Place the mouse on the electric blanket to keep it warm until it wakes up. 
	NOTE: The mouse should be monitored until it regains sufficient consciousness to maintain sternal recumbency. Postoperative exposure to bright light should be minimized.</li>
	<li>Remove the 100 D contact lens.</li>
	<li>Apply the levofloxacin eye gel to protect the cornea.</li>
	<li>Place the mouse back in the cage after it wakes up. 
	NOTE: Ensure that the examined mouse is not returned to the company of other mice until fully recovered.</li>
	<li>Turn off the software and switch off the computer.</li>
	<li>Clean the 100 D contact lens with water; dry the lens.</li>
	<li>Clean and disinfect the environment.</li>
</ol>
10. Image analysis
<ol>
	<li>Compare the OCT images of Vldlr knockout mice with those of C57BL/6J mice.</li>
	<li>Observe multiple positions: vertical and horizontal scans passing through the optic papilla; superior, inferior, nasal, and temporal scans; and abnormal reflection site scans.</li>
	<li>Observe the thickness, shape, layering, and abnormal reflectance lesions of the retina in each image, as well as the vitreous interface of the retina and the vitreous body.</li>
	<li>Record the locations, characteristics, and numbers of lesions.</li>
</ol>
11. Retinal stratification correction
<ol>
	<li>Click Load Examination on the OCT interface (Figure 5A).</li>
	<li>Call out the OCT images of a mouse from a pop-up window.</li>
	<li>Select images: OCT image scanning through the optic papilla, horizontally or vertically.</li>
	<li>Double-click the image in the Media Container to display it on the screen (Figure 5C).</li>
	<li>Click on Layer Detection to complete automatic layering on the retina (Figure 5D).</li>
	<li>Select the dividing lines on both sides of the layer prepared for analysis (Figure 6D-10).</li>
	<li>Select a separate dividing line (Figure 6B-6) and click Edit Layer (Figure 6A-1) to activate the line when a red circle appears (Figure 6B-7).</li>
	<li>Adjust Spacing (Figure 6A-4, e.g., 50) and Limit Range (Figure 6A-5, e.g., 50).</li>
	<li>Modify the dividing line by moving the red circle (compare the green dividing line in Figure 6B and Figure 6C; Figure 6C shows the modified result).</li>
</ol>
12. Retinal lamination thickness
<ol>
	<li>Click the Measure Marker button (Figure 6D-9).</li>
	<li>Select the dividing line of the layer to be analyzed (e.g., in the outer nuclear layer, select the 4th and 5th dividing line in the list) to display the boundary of the layer on the OCT image (Figure 6D-10).</li>
	<li>Select Connect with Layer (Figure 6D-11) and Stay Connected on Move (Figure 6D-12).</li>
	<li>Select the area to display the results (the selected column is colored, Figure 6D-13).</li>
	<li>Click the position to be analyzed on the OCT image to make the measurement line appear (perpendicular to the horizontal axis and consistent with the color of the resulting area) (Figure 6D-14).</li>
	<li>Click on the next column for the next measurement and reveal the previous data (Figure 6E-15).</li>
	<li>Read the Vert value (thickness of the measured position) in the Length in &#181;m (tissue) row (Figure 6E, red rectangle).</li>
	<li>Click Delete Marker (Figure 6E-16) and New Marker (Figure 6E-17) to retest so that the results will cover the original data (if remeasurement is necessary).</li>
	<li>Press Print Scr on the keyboard to save screenshots, or click Save Examination to save directly (Figure 5H).</li>
	<li>Input the data into a spreadsheet or statistical software for statistical analysis.</li>
</ol>
13. Measurement of full retinal thickness
<ol>
	<li>Select line 1 (ILM, inner limiting membrane, Figure 7B) and line 7 (OS-RPE, OS: outer photoreceptor segments; RPE: retinal pigment epithelial layer, Figure 7C) in the list in the upper right corner. 
	NOTE: The full retinal thickness means the thickness of the retinal neurepithelium layer, which is the retina between ILM and OS-RPE on OCT).</li>
	<li>Measure the retinal thickness on both sides of the optic papilla at a specific interval.
	<ol>
		<li>For example: from the appearance of the retinal structure at the edge of the optic papilla, measure 4 values with 200 &#181;m spacing of the horizontal ruler (Figure 7G, H).</li>
	</ol>
	</li>
	<li>Record all measured values in a spreadsheet.</li>
	<li>Use multiple t-tests (one per row) to compare the measured values of each corresponding position in both groups.</li>
</ol>

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The authors declare no potential conflict of interest.

In this study, OCT imaging using a small-animal retinal imaging system was applied to evaluate retinal changes in Vldlr knockout mice, which demonstrate incomplete posterior vitreous detachment, subretinal neovascularization, and retinal thickness thinning. OCT is a noninvasive imaging method to examine the condition of the retina in vivo. Most OCT devices are designed for human eye examination. The size of the hardware equipment, the setting of the focal length, the setting of the system parameters, an...

Optical coherence tomography (OCT) is an imaging technique that can provide in vivo high resolution and crosssectional imaging for tissue1,2,3,4,5,6,7,8, especially for the noninvasive examination in the retina9,10,11,12. It can also be used to quantify some important biomarkers, such as retinal thickness and retinal nerve fiber layer thickness. The principle of OCT is optical coherence reflectometry, which obtains crosssectional tissue information from the coherence of light reflected from a sample and converts it into a graphic or digital form through a computer system7. OCT is widely used in ophthalmology clinics as an essential tool for diagnosis, follow-up, and management for patients with retinal disorders. It can also provide insight into the pathogenesis of retinal diseases.
In addition to clinical applications, OCT has also been used in animal studies. Although pathology is the gold standard of morphological characterization, OCT has the advantage of noninvasive in vivo imaging and longitudinal follow-up. Furthermore, it has been shown that OCT is well correlated with histopathology in retinopathy animal models11,13,14,15,16,17,18,19,20. The mouse is the most commonly used animal in biomedical studies. However, its small eyeballs pose a technical challenge to conducting OCT imaging in mice.
Compared to the OCT first used for retinal imaging in mice21,22, OCT in small animals has now been optimized with respect to hardware and software systems. For example, OCT, in combination with the tracker, significantly reduces the signal-to-noise ratio; OCT software system upgrades allow more retinal layers to be detected automatically; and the integrated DLP beamer helps to reduce the motion artifacts.
Very-low-density lipoprotein receptor (Vldlr) is a transmembrane protein in endothelial cells. It is expressed on retinal vascular endothelial cells, retinal pigment epithelial cells, and around the outer limiting membrane23,24. Subretinal neovascularization is the phenotype of Vldlr knockout mice23. Therefore, Vldlr knockout mice are used to investigate the pathogenesis and potential therapy of subretinal neovascularization. This article demonstrates the application of OCT imaging to detect retinal lesions in Vldlr knockout mice, hoping to provide some technical reference for retinopathy research in small animal models.

<table><tbody><tr><td>100-Dpt contact lens</td><td>Volk Optical,Inc, Mentor, OH</td><td></td><td>Accessory belonging to the RETImap</td></tr><tr><td>Double aspheric 60-Dpt glass lens</td><td>Volk Optical,Inc, Mentor, OH</td><td></td><td>Accessory belonging to the RETImap</td></tr><tr><td>Electric heating blanket</td><td>POPOCOLA</td><td>CW-DRT-01</td><td>50 x 35 cm</td></tr><tr><td>Injection syringe (1 mL)</td><td>Kaile</td><td></td><td>0.45 x 16RWLB</td></tr><tr><td>Levofloxacin Hydrochloride Eye Gel</td><td>EBE PHARMACEUTICAL Co.LTD</td><td></td><td>5 g: 0.015 g</td></tr><tr><td>Medical sodium hyaluronate gel</td><td>Alcon</td><td>16H01E</td><td></td></tr><tr><td>Microliter syringes</td><td>Shanghai high pigeon industry and trade co., LTD</td><td>Q31/0113000236C001-2017</td><td>50 &amp;micro;L</td></tr><tr><td>Povidone iodine solution</td><td>Guangdong medihealth pharmaceutical Co.,LTD</td><td></td><td>100 mL</td></tr><tr><td>RETImap</td><td>ROLAND CONSULT</td><td>19-99_50-2.1_1.2E</td><td>cSLO/ERG/VEP/FA/OCT/GFP</td></tr><tr><td>Small animal ear studs</td><td>OSMO POCKET OT110</td><td>INS1005-1S</td><td></td></tr><tr><td>Tropicamide Phenylephrine Eye Drops</td><td>Santen Pharmaceutical Co.,LTD</td><td></td><td>5 mg/mL</td></tr><tr><td>Xylazin</td><td>Sigma</td><td>X1251-5G</td><td>5 g</td></tr><tr><td>Zoletil 50</td><td>Virbac.S.A</td><td>7FRPA</td><td>Tiletamine 125 mg + Zolazepam 125 mg</td></tr></tbody></table>

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.

Thanks to the high-resolution scans of OCT, the layers of the mouse retina can be observed, and abnormal reflections and their exact locations can be identified. The retinal OCT images of Vldlr knockout mice and C57BL/6J mice were compared in this study. The OCT images of all C57BL/6J mice showed various retinal layers with different reflectivity, and the demarcation was clear (Figure 8D). In contrast, all Vldlr knockout mice showed abnormal, hyperreflective lesions on the ...

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Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

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 ...

application-optical-coherence-tomography-to-mouse-model

Research

JoVE Journal

Medicine

3.9K Views.  Shantou University and the Chinese University of Hong Kong. Optische coherentietomografie wordt veel gebruikt bij de klinische diagnose van verschillende retinale ziekten, maar het is nog steeds een uitdaging om toe te passen in muismodellen. Dit protocol beschrijft hoe de OCT-techniek bij dieren moet worden beheerd. Deze technologie kan worden toegepast op dieren met verschillende oogbolgroottes.Het maakt niet-invasieve, hoge resoluties en real-time retinale cross-sectionele beeldvorming mogelijk, samen met kwantitatieve metingen van retinale ...

Video: Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

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 ...

Neuroscience

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

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into 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 ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

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 ...

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 ...

In Vivo Imaging of the Mouse Retina Using Optical Coherence Tomography

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/55865/optical-coherence-tomography-imaging-mouse-retinal-ganglion-cells-in-vivo'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Jagodzinska, J.,&#160;et. al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>,&#160; Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells&#160;In Vivo<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>.&#160;J. Vis. Exp.&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>(2017)</a>&#160; &#160;In this video, we demonstrate the use of spectral-domain optical coherence tomography (SD-OCT) to image retinal layers and assess structural changes in a genetically modified mouse model with optic neuropathy.

Source:&#160;Jagodzinska, J.,&#160;et. al.,&#160; Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells&#160;In Vivo.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Optical Coherence Tomography Imaging for Assessing Uveitis in a Mouse Model

After anesthetizing the mouse, dilate the pupil with one drop of 2.5% phenylephrine, avoiding excess droplets entering the nose or mouth. After two to three minutes, dab off the excess liquid, and cover the eye with 0.3% hypromellose gel. Next, wrap the mouse in a layer of surgical gauze to maintain body warmth, then place the mouse on the animal cassette, and position the head with the bite bar.
To acquire the optical coherence tomography, or OCT images, turn on the OCT imaging system, and open the imaging software. For posterior chamber imaging, bring the OCT close to the eye&#39;s surface, taking care not to bring the surface of the lens in contact with the eye.
Once the eye is correctly positioned, stop the Fast Scan, select the Volume Scan protocol, and activate the scan with the Aim option. For posterior segment images, adjust until the optic nerve is centered in the horizontal B-Scan alignment image and the retina is aligned with the vertical alignment axis. For anterior segment images, adjust the position to center the apex of the cornea in both the horizontal B-Scan alignment image and vertical B-Scan alignment image. Finally, click on Snapshot to capture the volume scan image. Then click on Save.

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

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