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

摘要

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

摘要

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.

引言

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 ....

研究方案

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 solutio....

结果

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.......

讨论

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.......

材料

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 Antisedane)	Orion	Vnr 47 19 53	Anesthesia