Vascular Parameters of Normal Cynomolgus Macaques by Fundus Fluorescence Angiography and Optical Coherence Tomography Angiography

Background: To provide normal vascular parameters for cynomolgus macaques. Compare the advantages and disadvantages of Fundus fluorescence angiography (FFA) and Optical coherence tomography angiography (OCT-A) in angiography. To establish an eye parameter database for cynomolgus macaques. Methods: Five normal cynomolgus macaques were studied for the collection of data, with a mean age of 4.6±0.55 years. The Heldelberg Spectralis® HRA+OCT was used to obtain parameters for Fundus fluorescence angiography (FFA). The vessel density was measured using the RTVue XR with AngioVue (software version 2017.1.0.155; Optovue, Inc., Fremont, CA, USA); the scan sizes of the macular and optic discs were 3×3mm and 4.5×4.5mm, respectively. Results: Cynomolgus macaque's fundus fluorescence angiography had similar stages as those found in humans. Optical coherence tomography can image the superficial, deep capillary plexus and the radial peripapillary capillary network. The highest whole En-face mean vessel density (VD) in the macular area was 68.19±0.75 % in the choroid capillary layer. In both layers of the optic disc, the vessel density in the Nasal quadrant was lower than in the inferior-tempo quadrant. Conclusions: This is a rare research, where we have collected the normal FFA and the novel OCT-A parameters for cynomolgus macaques. This study provides normal vascular parameters of cynomolgus macaques via Fundus fluorescence angiography (FFA) and Optical coherence tomography angiography (OCT-A), helping to establish an optical parameter database for cynomolgus macaques, promoting choroid-retinopathy research. Background A, et NEW INSIGHT INTO THE MACULAR DEEP VASCULAR PLEXUS IMAGED BY OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY. Retina


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Retina plays a vital role in vision. The metabolic activity of retina is higher than other human tissues1,2 and therefore the vascular circulation of retina is complex. A variety of techniques have been used to measure retinal perfusion. Fundus fluorescence angiography (FFA) was routinely used to evaluate retinal vascular retinopathy3 as it could analyze the choroid-retinal vasculature, and show vascular leakage and neovascularization4. However, there are some limitations to this methodology, such as the intravenous injection of contrast agents that can lead to some side-effects5,6.
Additionally, FFA images are unable to extract further details of deeper capillary plexus, due to limited depth perception. Optical coherence tomography angiography (OCT-A) is a novel imaging method7, that detects the flow of blood via intrinsic signal, without the requirement of using an intravenous agent. It can visualize and divide the retina into various layers, clearly showing the capillary vessels in each layer. The ability of OCT-A to quantify blood flow and vascular density of the retinal choroidal circulation is essential to the study of global ophthalmology.
Owing to close similarity to humans in optical structure and function, nonhuman primates like cynomolgus macaque serve a crucial role in ophthalmic disease research8-11. The FFA and OCTA vascular parameters for normal humans have been reported before1,12, and researchers have reported the flow perfusion parameters for normal monkeys13. However, there is very little literature available on the vascular density parameters for the macular and optic disc of ocularly normal cynomolgus macaques. Our study aims to evaluate the macular vascular circulation parameters in normal rhesus monkeys using OCTA and FFA, and to compare the advantages and disadvantages of the two techniques in angiography.

Animals
Data was collected from five normal adult male cynomolgus macaques, with a mean age of 4 4.6±0.55 years, and a mean weight of 4.44±0.88 kg. The animal production license number was SCXK(YUE)2014-0027 (GuangZhou Blooming-Spring Biological Technology Development Co. Ltd). Before the start of the experiments, all five animals were determined to be ocularly normal and healthy. After these experiments, the animals are in good care and will be used for further research.

Preparation of animal
Animals were anesthetized with an intramuscular Injection (im.) of ZoletilTM50 (VIRBAC S.A.), which is the combination of Tiletamine-Zolazepam (4mg/kg). Anesthesia was maintained during examinations by intravenously injecting supplemental doses of ZoletilTM50 (1/3 of the initial dose) as needed. Body temperature was monitored using a water circulating heating pad at 37℃. Pupils were fully dilated to approximately 9 mm in diameter with Tropicamide Phenylephrine Eye Drops (0.5%, Santen-China, China). Sodium Hyaluronate Eye Drops (0.3%, Santen-China, China) were used to maintain corneal moisture. A restraining device was used to maintain stable positioning of the animal's eyes and head. The eyelids were opened with a lid speculum. The test images acquired by FFA and OCT-A were loaded onto Photoshop CS6. The FFA images for the macular and optic disc areas were enlarged and intercepted to match the details seen on the optical coherence tomography angiography images. IBM SPSS statistics version 19.0 (SPSS, Inc. Chicago, USA) was used to analyze the data. The measurements are presented as mean ± standard deviation (SD). The vessel density comparison of different layers and different regions of each layer were analyzed via Bonferroni analysis. The significance adopted was, P < 0.05.

Stages of Fundus fluorescence angiography
Human Fundus fluorescence angiography is mainly divided into five stages: early arterial stage, arterial stage, arteriovenous stage, venous stage and later stage. In our study, cynomolgus macaques' FFA images tend to have similarities with the human angiographic stages. As seen in Figure 1 Researchers can quantitatively analyze vessel parameters via high-resolution imaging in OCT-A. Therefore in this study, we further compared the vessel density of macular, optic disc and surrounding regions via OCT-A. There are four levels of choroid-retinal capillary networks in the macular of cynomolgus macaques, including the superficial layer, the deep retina layer, the outer retina layer, and the choroid-capillary. Comparing the whole En-face vessel density among the four layers, the highest was found to be in the Choroid Cap layer.
These results are in concordance with Florence Coscas' research in humans1. One possible explanation for this maybe that the deep layer is formed by a homogenous capillary vortex21, whereas the superficial layer is formed only by transverse capillaries; additionally, the superficial layer may artificially influence the vessel density assessment of the DCP1. In the fovea area, the vessel density is the lowest, compared to the other sections of the retinal layer, probably due to the fovea avascular zone (FAZ)18.
We found the vessel density of the whole En-face and the Inside Disc section to be higher in the nerve head layer. In contrast, the VD of peripapillary region is much higher in radial peripapillary capillary layer. In both of the optic disc layers, the vessel density in nasal quadrant is lower than inferior-tempo quadrant. Overall, optical coherence tomography can image the radial peripapillary capillary network better. The blood supply associated with early optic disc lesions in glaucoma patients comes from the microcirculation of the posterior ciliary artery, hence the observation of the radial peripapillary capillary layer is beneficial in the early diagnosis of glaucoma22.
Despite the novelty of OCT-A, especially in assessing different diseases23-27, it's still unclear how OCT-A may be used in disease management, especially in animal models.
Nowadays, the nonhuman primates, especially cynomolgus macaques, serve a very important role in the research of ophthalmic diseases owing to similarities in optical structure and function with humans8-11. Therefore, fully understanding the traditional FFA and the novel OCT-A parameters and comparing their images can help in establishing these techniques, especially OCT-A, for the diagnosis of disease in animal models.
In conclusion, we evaluated the macular vascular circulation parameters in normal cynomolgus macaques by OCT-A and FFA and compared the advantages and disadvantages of the two techniques in angiography. Furthermore, we analyzed the vessel density parameters of the choroid-retinal vascular. All in all, our research provides normal vascular parameters of cynomolgus macaques, promoting the establishment of an eye parameter database for non-human primates. Our study does have certain limitations that need to be addressed. Such as the experimental animal used was cynomolgus macaque, and therefore its unclear if these results are applicable to other species. Our future goal is to do an extensive study across different species, comparing the similarities and differences of FFA Date availability statements: The datasets generated and analysed during the current study are not available due to that we have further research related to the subject, but are available from the corresponding author on reasonable request. Tables   Table 1     Supplementary Files