In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Podsumowanie

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Streszczenie

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.

The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch's membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch's membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.

This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

Wprowadzenie

The first successful attempt to mimic the pathology of human CNV in rodents was demonstrated almost three decades ago with a krypton laser in Long-Evans rats¹. Thereafter, a krypton laser was used to break Bruch's membrane in the most popular mouse strain, C57BL/6J^2,3,4. The success rate of CNV induction was verified with FA and histological stains. A rapid development of noninvasive imaging modalities, such as OCT, fostered the growth of the field of rodent preclinical models. The ability to monitor morphological changes in the retina at multiple time points in the same eye significantly contributes to the reduction of animal use, and increases efficiency in experimental studies. Histological evaluation of CNV lesions is rather straightforward, and requires labeling of abnormal vascular growth around the site of laser administration, image acquisition, and area/volume estimation using an image analysis software. In contrast, in vivo imaging modalities introduce more complex analyses of CNV pathology and its interpretation.

Here we present a simple and relatively fast method to grade induction, progression, and regression of CNV using FA, SD-OCT, and the automated segmentation method in the mouse laser-induced CNV model.

Protokół

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the EC Directive 86/609/EEC for animal experiments, using protocols approved and monitored by the Animal Experiment Board of Finland.

1. Laser-induced mouse CNV model ⁵

1. Inspect the eyes of the animal macroscopically for any abnormalities.
2. Weigh the mouse.
3. Calculate and prepare an appropriate amount of anesthetics to use, based on the weight of the animal, e.g. a mixture of medetomidine (1 mg/kg), ketamine (75 mg/kg), and distilled water (0.9% NaCl solution) at a ratio of 1:1.5:2.5, or ketamine (40-75 mg/kg), xylazine (5 mg/kg), and distilled water (0.9% NaCl solution) at a ratio of 1:2.5:1; for a 20 g mouse, inject 0.1 mL of mixture.
4. Inject anesthetic intraperitoneally.
5. Place mouse back into the cage and wait until animal is anesthetized. Confirm the mouse is properly anesthetized by lack of a pedal reflex.
6. Ensure the use of laser safety personal protective equipment.
7. Turn on a slit lamp and a 532 nm diode laser.
8. Remove the mouse from the cage and place on the heating pad.
9. Apply one drop of Tropicamide for pupillary dilation. Wait for 3-5 min for full (3 mm) pupillary dilation.
10. Place the mouse on the stage of the slit lamp.
11. Place one drop of opthalmic liquid gel on a coverslip to applanate the cornea.
12. Orient the mouse eye with the optic nerve head in the center.
13. Set the laser power to 100 mW, the duration to 100 ms, and the spot size to 50 µm.
14. Focus the laser beam on the retinal pigment epithelium (RPE).
15. Make three laser shots into one eye by avoiding retinal blood vessels ideally at the 4, 8, and 12 o'clock positions around the optic nerve, respectively. Inspect the fundus of the eye after all laser shots for absence of retinal bleeding. The contralateral eye serves as a non-lasered control.
16. Discard the coverslip and place mouse back on the heating pad.
17. Apply one drop of PEG gel drops on both eyes.

2. SD-OCT ^6,7

1. Place the mouse into the rodent alignment stage, and immobilize the head.
2. Align the lens of the SD-OCT system (e.g., Bioptigen/Leica Envisu R2200) to face the eye for in vivo imaging using X- and Y-stage controllers.
3. Perform SD-OCT scans to verify breaks of Bruch's membrane: once the SD-OCT scans the whole eye, manually move the reference line on the lasered sites. Breaks of Bruch's membrane should be clearly visible in lasered areas (see Figure 1).

3. Fluorescein angiography ^7,8,9

1. Remove mouse with the holder, and place it on the FA system (e.g., Heidelberg Spectralis HRA2).
2. Focus at laser burn areas of the fundus of the eye using infrared reflectance mode with the head of the optic nerve in the middle of the viewing window.
3. Inject 0.1 mL of 5% fluorescein sodium salt for a 20 g mouse subcutaneously or intraperitoneally.
4. Focus on the choroidal level.
5. Take an image from the choroidal focus level.
6. Re-focus at the retinal level and take an image.
7. Wait for 30 s and repeat steps 3.4-3.6.
8. Remove the mouse from the holder and place it on the heating pad.
9. Reverse anesthesia by α2-antagonist for medetomidine, atipamezole (0.5 mg/kg, i.p.), or wait for animal recovery from anesthesia.
10. Repeat in vivo SD-OCT and FA imaging in anesthetized animals on the follow-up days 5, 10, and 14.

4. CNV Grading

1. Grade the damage of Bruch's membrane from OCT images and choroidal FA images taken immediately after the lasering on day 0 as following:
   0 - Bruch's membrane was not damaged
   1 - successful damage of Bruch's membrane
2. Grade the presence of CNV from lasered spots that had leakage as observed by comparing dynamics of fluorescein signal in a series of retinal FA images as following:
   0 - normal appearance of retina
   0.5 - faint staining of leakage
   1.0 - leaky CNV areas
   Note: Use the OCT imaging for additional confirmation of CNV or in questionable FA where the presence of intraretinal fluid in OCT images would suggest CNV grading.

5. Retinal Thickness Measurements

1. Use an automated segmentation software for retinal thickness measurements. Ensure that the total retinal thickness is considered, as the thickness of all layers from nerve fiber layer to RPE (healthy measurement sites), or to an imaginary line connecting RPE around the site of damage (lasered sites) (see also Figure 7).

Wyniki

A bubble or subretinal bleeding immediately after lasering is not always visible. Therefore, SD-OCT is particularly important to verify damage of Bruch's membrane. Figure 1 shows an example of OCT imaging at different time points after laser administration.

Figure 1: OCT...

Dyskusje

Multimodal imaging offers valuable tools for CNV pathology evaluation. Here we presented an imaging protocol consisting of FA, SD-OCT, and automatic segmentation for the quick, reproducible, and reliable evaluation of CNV pathology. A break of Bruch's membrane after laser administration was confirmed. In addition, the use of SD-OCT at this stage also allowed immediate visualization of possible intraretinal and subretinal hemorrhages, which may confound the interpretation of results. Retinal leaks were graded based on the...

Materiały

Name	Company	Catalog Number	Comments
Medetomidine (commercial name Domitor)	Orion	Vnr 01 56 02	Anesthesia
Ketamine	Intervet	Vnr 51 14 85	Anesthesia
0,9% NaCl	B Braun	357 0340	Anesthesia
Xylazine (commercial name Rompun vet)	Bayer	vnr 14 89 99	Anesthesia
Tropicamide	Santen	Vnr 04 12 36	Mydriatic agent
Viscotears	Alcon	Vnr 44 54 81	Lubricant
Systane	Alcon	-	Lubricant
5% Fluorescein sodium salt	Sigma Aldrich	F6377-100G	Fluoresent agent
Atipamezole (commercial name Antisedan)	Orion	Vnr 47 19 53	Anesthesia