Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove

Medicine

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Published: January 12, 2022 doi: 10.3791/63421
* These authors contributed equally

Summary

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

Abstract

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.

Introduction

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.

Subscription Required. Please recommend JoVE to your librarian.

Protocol

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. Two-month-old C57/BL/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

  1. Assign the mice to two groups: an experimental group consisting of Vldlr knockout mice and a control group consisting of C57BL/6J mice.
  2. Feed the mice with food and water conventionally.
  3. Raise the mice in the animal laboratory under stable conditions of room temperature (22 °C), humidity (50-60%), light-dark cycle (12 h-12 h), and room light intensity (350-400 lux).
  4. Prepare the experimental equipment: optical coherence tomography with confocal scanning laser ophthalmoscope (cSLO) for small animals (Figure 1A).
  5. Prepare all materials required for the experiment (Figure 1B) and weigh the mice (Figure 1C).

2. Anesthetic configuration

  1. Add 3.57 mL of sterilized water to a bottle of lyophilized anesthetic powder (Tiletamine 125 mg + Zolazepam 125 mg) to achieve a final concentration of 70 mg/mL.
  2. Dissolve 200 mg of Xylazine powder in 10 mL of sterilized water to a final concentration of 20 mg/mL.
  3. Mix the two solutions in a new tube (take the same volume to mix, volume ratio 1:1);
  4. Set the injection dosage of the anesthetic mixture solution as 1 µL/g.
    ​NOTE: Dosage selection should be according to the experiment duration. When the dosage is 1 µL/g (1 µL of anesthetic mixture for each 1 g weight of mouse), the duration of deep anesthesia is ~1 h. When the dosage is 1.5 µL/g (1.5 µL of anesthetic mixture for each 1 g weight of mice), the duration of deep anesthesia is ~2 h. The dosage of 1 µL/g anesthetic mixture met the time requirement of OCT (0.5-1 h) for one mouse.

3. Information records

  1. Record the information: group, code, date of birth, age, sex, weight, and anesthetic dosage.

4. Instrument startup and testing

  1. Switch on the computer and start up the software.
  2. Click the Test program button to complete the test program.
  3. Turn on the thermostat and preheat it to the temperature of 37 °C.
  4. Start the OCT module procedure after the program testing.
  5. Create a new subject and fill in the mouse information.
  6. Preheat the electric blanket and cover it with surgical towels.

5. Application of mydriatic drops

  1. Grab the mouse, pull the neck fur, make the eyeball protrude slightly, and rotate the mouse head with one eye facing upward.
  2. Apply the mydriatic drops to the eyes to dilate the pupil, one drop in each eye (Figure 2A).
  3. Rotate the head to add the mydriatic eye drop to the other eye.
  4. Wipe away any excess liquid spilling over the face.
  5. Check for pupil dilation after 10 min.

6. Anesthesia: Intramuscular injection (after pupil dilation)

  1. Calculate the injection volume of the anesthetic mixture solution according to the weight, e.g., 20 µL/20 g (Figure 1C).
  2. Extract the corresponding volume of the anesthetic mixture with a 50 µL microsyringe, e.g., 20 µL.
  3. Select the lateral thigh muscles of the mouse as the injection point.
  4. Remove the localized hair with scissors to expose the thigh muscles.
  5. Disinfect the skin three times with povidone-iodine (Figure 1B-I and Figure 2B).
  6. Insert the microsyringe needle into the muscle at an angle of 60-90° with the bevel of the needle mouth facing upwards and a depth of ~0.3 cm (ensure that the bevel is fully inserted into the muscle; Figure 2C).
  7. Inject all of the anesthetic solution in the microsyringe (Figure 2C).
    NOTE: A smooth injection with no spillover indicates successful injection.
  8. Pull out the needle quickly after injection.
  9. Press the injection area with a cotton swab for 30 s.
  10. Ensure that there is no local bleeding and congestion.

7. Placement of the mouse

  1. Place a mouse on an electric blanket platform.
  2. Coat both eyes with medical sodium hyaluronate gel immediately after anesthesia (Figure 2D).
  3. Screw a 60 D double spherical lens (preset lens) on the cSLO device (Figure 1A-5,6).
  4. Place a 100 D contact lens on the mouse cornea with the concave side touching the sodium hyaluronate gel on the corneal surface (Figure 2E,F and Figure 3A-II).
  5. 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).
  6. Adjust the angle of the contact lens with forceps to keep the pupil in the center of the lens.
  7. Fine-tune the adjustments to the head to make the eye face straight ahead.

8. Confocal Scanning Laser Ophthalmoscope (cSLO)

  1. Click the OCT button, choose the mouse module, and start the cSLO program (Figure 4B).
  2. Select the IR mode (light source: red light), and adjust the parameter (range: 2047, Figure 4D).
  3. Select the eye to be examined (right eye: Figure 4C-1; left eye: Figure 4C-2)
  4. Control the lever and move the preset lens towards the contact lens slowly.
  5. Adjust the diopter value until the posterior pole imaging is clear (Figure 4E).
  6. Make further adjustments to align the image of the retinal posterior pole, centering it at the optic nerve head.

9. Optical coherence tomography (OCT)

  1. Start the OCT program (Figure 4G).
  2. Click the progress bar up and down until the OCT image appears (Figure 4H).
  3. Adjust parameters: Range Min (Figure 4I) = 0-20, Range Max (Figure 4J) = 40-60.
  4. Adjust the preset lens distance and position direction until an ideal OCT image is obtained.
  5. Select the scanning position by moving the standard line in the cSLO (Figure 4M).
  6. Start scanning from the optic nerve head.
  7. Collect images in the same order for each eye: horizontal line: optic nerve head → superior → inferior; vertical line: optic nerve head → nasal → temporal.
  8. Collect images from four directions.
  9. Click Average to overlay the cSLO and OCT image signals (Figure 4F and Figure 4O).
  10. Click the shot button to acquire the SLO-OCT image (Figure 4P).
  11. Save and export all the images (Figure 4Q, R).

10. The end of the experiment (after the OCT examination)

  1. 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.
  2. Remove the 100 D contact lens.
  3. Apply the levofloxacin eye gel to protect the cornea.
  4. 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.
  5. Turn off the software and switch off the computer.
  6. Clean the 100 D contact lens with water; dry the lens.
  7. Clean and disinfect the environment.

11. Image analysis

  1. Compare the OCT images of Vldlr knockout mice with those of C57BL/6J mice.
  2. Observe multiple positions: vertical and horizontal scans passing through the optic papilla; superior, inferior, nasal, and temporal scans; and abnormal reflection site scans.
  3. 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.
  4. Record the locations, characteristics, and numbers of lesions.

12. Retinal stratification correction

  1. Click Load Examination on the OCT interface (Figure 5A).
  2. Call out the OCT images of a mouse from a pop-up window.
  3. Select images: OCT image scanning through the optic papilla, horizontally or vertically.
  4. Double-click the image in the Media Container to display it on the screen (Figure 5C).
  5. Click on Layer Detection to complete automatic layering on the retina (Figure 5D).
  6. Select the dividing lines on both sides of the layer prepared for analysis(Figure 6D-10).
  7. Select a separate dividing line (Figure 6B-6) and click Edit Layer (Figure 6A-1) to activate the line when a redcircle appears (Figure 6B-7).
  8. Adjust Spacing (Figure 6A-4, e.g., 50) and Limit Range (Figure 6A-5, e.g., 50).
  9. 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).

13. Retinal lamination thickness

  1. Click the Measure Marker button (Figure 6D-9).
  2. Select the dividing line of the layer to be analyzed (e.g., in the outer nuclearlayer, select the 4th and 5th dividing line in the list) to display the boundary of the layer on the OCTimage (Figure 6D-10).
  3. Select Connect with Layer (Figure 6D-11) and Stay Connected on Move (Figure 6D-12).
  4. Select the area to display the results (the selected column is colored, Figure 6D-13).
  5. 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).
  6. Click on the next column for the next measurement and reveal the previous data (Figure 6E-15).
  7. Read the Vert value (thickness of the measured position) in the Length in µm (tissue) row (Figure 6E, red rectangle).
  8. 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).
  9. Press Print Scr on the keyboard to save screenshots, or click Save Examination to save directly (Figure 5H).
  10. Input the data into a spreadsheet or statistical software for statistical analysis.

14. Measurement of full retinal thickness

  1. 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).
  2. Measure the retinal thickness on both sides of the optic papilla at a specific interval.
    1. For example: from the appearance of the retinal structure at the edge of the optic papilla,measure 4 values with 200 µm spacing of the horizontal ruler (Figure 7G,H).
  3. Record all measured values in a spreadsheet.
  4. Use multiple t-tests (one per row) to compare the measured values of each corresponding position in both groups.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

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 OCT images (Figure 8B).

Incomplete vitreous detachment (PVD) in Vldlr knockout mice
The OCT results showed some middle reflective bands on the retinal surfaces of Vldlr knockout mice (Figure 8B, red arrows). These middle reflective bands adhered to the retinal vessel (Figure 8B, green arrow), corresponding to the cSLO image (Figure 8A, green arrow). These features are consistent with the OCT characteristics of incomplete vitreous detachment.

Subretinal neovascularization in Vldlr knockout mice
The results showed that subretinal neovascularization had two development modes in the Vldlr knockout mice.

With involvement of the outer nuclear layer
A hyperreflective lesion, with a bottom-down triangular shape on the OCT image, appeared on the subretinal space and spread to the outer nuclear layer. The lesion did not break through the outer plexiform layer (Figure 8B, white arrow).

The OCT appearance of this type of subretinal neovascularization was consistent with the pathological findings shown in Figure 9A. The pathological section showed that neovascularization (Figure 9A, thick green arrow) broke through the RPE, photoreceptor inner/outer segments (IS/OS), and the external limiting membrane (ELM). It invaded the outer nuclear layer (ONL) but did not break through the outer plexiform layer (OPL).

Without involvement of the outer nuclear layer
A band of hyperreflective lesion appeared on the OCT image, which was located at the subretinal space (Figure 8B, yellow arrow). The cSLO image showed the corresponding location (Figure 8A, yellow arrow). The additional scans of the retina around this location (Figure 8A, yellow arrow) showed the same findings.

Consistent with the lesion (Figure 10A, thick blue arrow) in the pathological section, this subretinal neovascularization did not break through the ELM (Figure 10A, thin yellow arrow) but partially involved the photoreceptor IS/OS.

Retinal thickness results
The retinal thickness of the right eye of all mice was obtained by using the automatic stratification and thickness measurement function of OCT. The retinal thickness of Vldlr knockout mice (200.94 ± 14.64 µm) was significantly lower than that of C57BL/6J mice (217.46 ± 10.21 µm, P < 0.001, t-test, 7 right eyes/group). The comparison of retinal thickness in the four directions (temporal, nasal, superior, and inferior) of the posterior polar between the two groups is shown in Figure 11.

Figure 1
Figure 1: Preparation of experimental materials and animals. (A) Equipment: 1. cSLO/OCT device for small-animal retinal imaging, 2. computer and monitor, 3. Small, constant-temperature animal platform, 4. thermostat, 5. preset lens, 6. installation of the preset lens. (B) Medicines and small items: I. povidone-iodine, II. microsyringe, III. anesthetic mixture solution, IV. timer, V. mydriatic eye drops, VI. forceps, VII. medical sodium hyaluronate gel, VIII. medical cotton swab, IX. antibiotic eye ointment, X. 100 D contact lens (two). (C) Weight measurement on a digital balance. Abbreviations: cSLO = confocal scanning laser ophthalmoscope; OCT = optical coherence tomography. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Preparation before OCT examination of mice. (A) Mydriasis eye drop application, (B) disinfection at the anesthetic injection site with povidone-iodine, (C) intramuscular injection of the anesthetic drug, (D) sodium hyaluronate gel coating on the cornea, (E, F) placement of a 100 D contact lens, with concave surface contacting the cornea. Abbreviation: OCT = optical coherence tomography. Please click here to view a larger version of this figure.

Figure 3
Figure 3: OCT examination procedures. (A) Mouse position placement, I. preset lens, II. contact lens, III. Small, constant-temperature animal platform. (B) Operation of the cSLO/OCT machine, IV. operating lever, V. tilt lever, VI. cSLO device. Abbreviations: cSLO = confocal scanning laser ophthalmoscope; OCT = optical coherence tomography. Please click here to view a larger version of this figure.

Figure 4
Figure 4: OCT imaging process. A. Measurement mode, B. Start Laser of the IR laser, C. eye selection (C-1-OD; C-2-OS), D. range of IR laser, E. the diopter, F. overlay of the cSLO image, G. OCT scanning start/stop laser button H. reference of OCT image, I. Range Min: 0-20, J. Range Max: 40-60, K. signal intensity of the image, L. scanning direction (e.g., vertical scan), M. scanning position selected by moving the green reference line (e.g., vertical scan through the optic papilla), N. real-time display of the OCT image, O. overlay of the OCT image, P. Shot: image acquisition, Q. SLO-OCT images that have been acquired, R. Save Examination: saving the examination result. Scale bars = 200 µm. Abbreviations: cSLO = confocal scanning laser ophthalmoscope; OCT = optical coherence tomography; IR = infrared; OD = right eye; OS = left eye. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Automatic retinal delamination interface on OCT system. A. Load Examination button, B. Media Container, showing all the OCT images, C. OCT image being selected for analysis, D. Layer Detection button for automatic retinal layering, E. dividing line list, F. automatic delamination on the retina, G. Edit Layer button for layered correction, H. Save Examination button for saving the results. Scale bars = 200 µm. Abbreviation: OCT = optical coherence tomography. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Layered correction (A-C) and thickness measurement (D-E). (A) Layered edit activation interface: 1. Edit Layer button, 2. dividing line list (e.g., selecting all lines), 3. activated dividing lines, 4. Spacing adjustment, 5. Limit Range adjustment. (B) Activation of a dividing line (e.g., line 3 in A), 6. line 3, the line between the inner plexiform layer and inner nuclear layer, 7. an example of layering error. (C) Layering error modification, 8. the red circle for modification. (D) An example of retinal lamellar thickness measurement, 9. Measure Marker button, 10. dividing lines of the outer nuclear layer, 11. Connect with Layer (the measurement will connect with the layer according to the dividing lines), 12. Stay Connected on Move (the measurement position is where the manual click stays), 13. the location of the result display, 14. the measurement line (perpendicular to the horizontal axis). (E) Measurement result acquisition, 15. the measurement results (red rectangle: Vert value is the thickness result), 16. Delete Marker button for measurement record deletion, 17. New Marker button for remeasurement (the new result will overwrite the original record). Scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Measurement of full retinal thickness. A. Measure Marker button, B. line 1 (ILM) and C. line 7 (OS-RPE) selection for showing the boundaries of the full-thickness retina, D. Connect with Layer selection, E. Stay Connected on Move selection, F. ruler bar (vertical and horizontal ruler bars, both 200 µm in length), G. measurement lines on the retina (4 lines with 200 µm of horizontal ruler length as spacing on each side of the optic papilla), H. the measurement results (the results are differentiated by different colors and correspond to the color of the measurement lines on the retina), I. Data extraction from the Vert value in the Length in µm (tissue) row. Scale bars = 200 µm. Abbreviations: ILM = inner limiting membrane; OS-RPE = the layer between photoreceptor outer segment and retinal pigment epithelium. Please click here to view a larger version of this figure.

Figure 8
Figure 8: Comparison of cSLO and OCT images of Vldlr knockout and C57BL/6J mice. cSLO (A) and OCT (B) images of Vldlr knockout mice compared with the cSLO (C) and OCT (D) images of C57BL/6J mice. Characteristics of OCT in Vldlr knockout mice (B): 1) Middle reflective line (B, red arrows) on the inner surface of the retina with adhesion to the retinal vessel (B, green arrow). 2) Hyperreflective lesions, located at the subretinal space, with (B, white arrow) or without (B, yellow arrow) involvement of outer nuclear layer. The arrows on the cSLO image (A) represent the locations of the corresponding color arrows on OCT image (B). Scale bars = 200 µm. Abbreviations: cSLO = confocal scanning laser ophthalmoscope; OCT = optical coherence tomography; Vldlr = very-low-density lipoprotein receptor. Please click here to view a larger version of this figure.

Figure 9
Figure 9: Mode 1: retinal paraffin sections with hematoxylin-eosin staining in Vldlr knockout and C57 BL/6J mouse. (A) An example of subretinal neovascularization invading the outer nuclear layer (thick green arrow), located in the middle part of the retina of a Vldlr knockout mouse. (B) Normal control, the middle part of the retina of a C57 BL/6J mouse. Scale bars = 50 µm. Abbreviations: Vldlr = very-low-density lipoprotein receptor; ILM = inner limiting membrane; NFL = retinal nerve fibre layer; GCL = retinal ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; ELM = external limiting membrane; IS = photoreceptor inner segment; OS = photoreceptor outer segment; RPE = retinal pigment epithelium layer. Please click here to view a larger version of this figure.

Figure 10
Figure 10: Mode 2: retinal paraffin sections with hematoxylin-eosin staining in Vldlr knockout and C57 BL/6J mouse. (A) An example of subretinal neovascularization without the involvement of outer nuclear layer (thick blue arrow) and with intact ELM (thin yellow arrow), located in the middle periphery retina in a Vldlr knockout mouse. (B) Normal control, the middle periphery retina of a C57 BL/6J mouse. Scale bars = 50 µm. Abbreviations: VLDR = very-low-density lipoprotein receptor; ILM = inner limiting membrane; NFL = retinal nerve fiber layer; GCL = retinal ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; ELM = external limiting membrane; IS = photoreceptor inner segment; OS = outer photoreceptor segment; RPE = retinal pigment epithelium layer. Please click here to view a larger version of this figure.

Figure 11
Figure 11: Comparison of retinal thickness between C57 BL/6J mice and Vldlr knockout mice (all data from the right eye). (A) Retinal thickness (µm) through the optic nerve papilla by OCT horizontal scanning. (B) Retinal thickness (µm) through the optic nerve papilla by OCT vertical scanning. The horizontal coordinate represents the measuring positions with spacing of 200 µm.*: P < 0.05, **: P < 0.01, ***: P < 0.001. Abbreviations: T = Temporal; P = Optic papilla; N = Nasal; S = Superior; I = Inferior; OCT = optical coherence tomography; VLDR = very-low-density lipoprotein receptor; OD = right eye. Please click here to view a larger version of this figure.

Subscription Required. Please recommend JoVE to your librarian.

Discussion

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, and the positioning requirements of the examinee are all based on the human eye. Modifications of the lens and system settings are required to examine small animals with human-specific OCT equipment. This paper presents small-animal OCT examination procedures.

The focal length is different during image scanning of different small animals with different sizes of eyeballs. This difference in focal length is critical and must be resolved to obtain clear and accurate fundus images. One effective method is replacing the objective lens with lenses of different curvatures. Due to its small eyeball, the mouse needs a contact lens of 100 D in front of the cornea in addition to the double-spherical 60 D preset lens of the OCT equipment.

The OCT can only provide line scans that only cover a limited region of the retina. Therefore, it is essential to standardize the protocol of OCT scans for qualitative and quantitative comparison of OCT findings in different groups. Three horizontal scans and three vertical scans were performed here. This machine provides a real-time cSLO image to monitor the location of the OCT scan so that the position of the scan can be adjusted accurately and conveniently. Additional scans can be added when an abnormal reflection is found.

The parameters of image acquisition need to be adjusted carefully. Here, it is recommended that the Range Min be 0-20 and the Range Max be 40-60 (Figure 4I,J). When the parameters are overadjusted, the signal contrast of the image would be enhanced, and the reflected signal of the retina with low reflection becomes lower or even black, and some morphological information will be lost.

The following are some tips to avoid image quality deterioration: 1. Place a contact lens in front of the eyes immediately after anesthesia to avoid cataracts; 2. Ensure that the preset lens and contact lens are clean; 3. Avoid hair entering between the cornea and the contact lens; 4. Ensure the doppler, contrast, and brightness in the OCT parameters are set appropriately.

The OCT images can be used to qualitatively detect lesions and quantitatively measure metrics such as retinal thickness. Here, a method is proposed to measure the retinal thickness at several locations, and the average can be calculated as the mean retinal thickness. This is achieved through the automatic stratification function of the OCT system. Therefore, the thickness of the retinal laminations can also be measured. The measurement method is simple and accurate (Figure 6 and Figure 7). The results showed that the retinal thickness was lower in Vldlr knockout mice than C57BL/6J mice, consistent with the literature25. The difference in retinal thickness between the two groups can be clearly shown by a graph generated from the measurements at multiple locations (Figure 11). Similar retinopathy analysis and retinal thickness measurement methods have also been reported in the Stargardt disease mouse model26. However, it is worth noting that the hyperreflective bands at the vitreous interface of the retina do not belong to the retinal tissue and should be removed during stratification. In addition, if subretinal lesions invade the retina, the thickness measurement should include the invaded portion.

This small-animal retinal imaging system has some limitations. For example, although it can provide clear images of the posterior pole within 35°, image acquisition of the peripheral retina is still challenging. In addition, cSLO forms a gray-scale image, which is not as good as a color fundus image to detect fundus lesions (pigmentation, bleeding, exudation). Hence, further improvements are needed. In summary, OCT examination by the cSLO machine could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors declare no potential conflict of interest.

Acknowledgments

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.

Materials

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

DOWNLOAD MATERIALS LIST

References

  1. Frombach, J., et al. Serine protease-mediated cutaneous inflammation: characterization of an ex vivo skin model for the assessment of dexamethasone-loaded core multishell-nanocarriers. Pharmaceutics. 12, (9), 862 (2020).
  2. Osiac, E., Săftoiu, A., Gheonea, D. I., Mandrila, I., Angelescu, R. Optical coherence tomography and Doppler optical coherence tomography in the gastrointestinal tract. Journal of Gastroenterology. 17, (1), 15-20 (2011).
  3. Xiong, Y. Q., et al. Diagnostic accuracy of optical coherence tomography for bladder cancer: A systematic review and meta-analysis. Photodiagnosis and Photodynamic Therapy. 27, 298-304 (2019).
  4. Andrews, P. M., et al. Optical coherence tomography of the aging kidney. & Clinical Transplantation. 14, (6), 617-622 (2016).
  5. Terashima, M., Kaneda, H., Suzuki, T. The role of optical coherence tomography in coronary intervention. The Korean Journal of Internal Medicine. 27, (1), 1-12 (2012).
  6. Avital, Y., Madar, A., Arnon, S., Koifman, E. Identification of coronary calcifications in optical coherence tomography imaging using deep learning. Scientific Reports. 11, (1), 11269 (2021).
  7. Huang, D., et al. Optical coherence tomography. Science. 254, (5035), 1178-1181 (1991).
  8. Tsai, T. H., et al. Optical coherence tomography in gastroenterology: a review and future outlook. Journal of Biomedical Optics. 22, (12), 1-17 (2017).
  9. Chen, J., et al. Relationship between optical intensity on optical coherence tomography and retinal ischemia in branch retinal vein occlusion. Scientific Reports. 8, (1), 9626 (2018).
  10. Chen, X., et al. Quantitative analysis of retinal layer optical intensities on three-dimensional optical coherence tomography. Investigative Opthalmology & Visual Science. 54, (10), 6846-6851 (2013).
  11. Cruz-Herranz, A., et al. Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis. Journal of Neuroinflammation. 16, (1), 203 (2019).
  12. Podoleanu, A. G. Optical coherence tomography. Journal of Microscopy. 247, (3), 209-219 (2012).
  13. Augustin, M., et al. Optical coherence tomography findings in the retinas of SOD1 knockout mice. Translational Vision Science & Technology. 9, (4), 15 (2020).
  14. Berger, A., et al. Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology. PLoS One. 9, (5), 96494 (2014).
  15. Burns, M. E., et al. New developments in murine imaging for assessing photoreceptor degeneration in vivo. Advances in Experimental Medicine & Biology. 854, 269-275 (2016).
  16. Jagodzinska, J., et al. Optical coherence tomography: imaging mouse retinal ganglion cells in vivo. Journal of Visualized Experiments: Jove. (127), e55865 (2017).
  17. Kocaoglu, O. P., et al. Simultaneous fundus imaging and optical coherence tomography of the mouse retina. Investigative Opthalmology & Visual Science. 48, (3), 1283-1289 (2007).
  18. Tode, J., et al. Thermal stimulation of the retina reduces Bruch's membrane thickness in age related macular degeneration mouse models. Translational Vision Science & Technology. 7, (3), 2 (2018).
  19. Wang, R., Jiang, C., Ma, J., Young, M. J. Monitoring morphological changes in the retina of rhodopsin-/- mice with spectral domain optical coherence tomography. Investigative Ophthalmology & Visual Science. 53, (7), 3967-3972 (2012).
  20. Xie, Y., et al. A spectral-domain optical coherence tomographic analysis of Rdh5-/- mice retina. PLoS ONE. 15, (4), 0231220 (2020).
  21. Li, Q., et al. Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse. Investigative Ophthalmology & Visual Science. 42, (12), 2981-2989 (2001).
  22. Horio, N., et al. Progressive change of optical coherence tomography scans in retinal degeneration slow mice. Archives of Ophthalmology. 119, (9), 1329-1332 (2001).
  23. Hu, W., et al. Expression of VLDLR in the retina and evolution of subretinal neovascularization in the knockout mouse model's retinal angiomatous proliferation. Investigative Opthalmology & Visual Science. 49, (1), 407-415 (2008).
  24. Wyne, K. Expression of the VLDL receptor in endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology. 16, (3), 407-415 (1996).
  25. Augustin, M., et al. In vivo characterization of spontaneous retinal neovascularization in the mouse eye by multifunctional optical coherence tomography. Investigative Opthalmology & Visual Science. 59, (5), 2054-2068 (2018).
  26. Fang, Y., et al. Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model. The FASEB Journal. 34, (3), 3693-3714 (2020).
This article has been published
Video Coming Soon
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Mai, X., Huang, S., Chen, W., Ng, T. K., Chen, H. Application of Optical Coherence Tomography to a Mouse Model of Retinopathy. J. Vis. Exp. (179), e63421, doi:10.3791/63421 (2022).More

Mai, X., Huang, S., Chen, W., Ng, T. K., Chen, H. Application of Optical Coherence Tomography to a Mouse Model of Retinopathy. J. Vis. Exp. (179), e63421, doi:10.3791/63421 (2022).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter