Vascular parameters of normal cynomolgus macaques and healthy humans obtained by fundus fluorescence angiography and optical coherence tomography angiography

Background: To provide and compare normal vascular parameters for cynomolgus macaques and healthy humans, compare the advantages and disadvantages of fundus fluorescence angiography (FFA) and optical coherence tomography angiography (OCT-A) in angiography, and establish an eye parameter database for cynomolgus macaques and healthy humans. Methods: 5 normal cynomolgus macaques with a mean age of 4.60±0.55 years were studied for data collection. A Heidelberg Spectralis® HRA+OCT was used to obtain parameters for FFA. The macula of 28 healthy humans (14 males and 14 females), with a mean age of 25.11±6.21 years and the optic discs of 9 healthy humans (4 males and 5 females) with a mean age of 28.56±6.78 years were measured. The vessel density (VD) was measured using an RTVue XR with AngioVue. The scan sizes of the macula and optic discs were 3×3 mm and 4.5×4.5 mm, respectively. Results: FFA of cynomolgus macaques revealed stages similar to those observed in humans. OCT-A was used to image the superficial and deep capillary plexuses and radial peripapillary capillary network. The highest entire en-face mean VD in the macular area was 68.19±0.75% in the choroid capillary layer. In both layers of the optic disc, the VD in the nasal quadrant was lower than the VD in the inferior temporal quadrant. Similarities and significant differences in VD between healthy humans and cynomolgus macaques were obtained using OCT-A. Conclusions: This study provides normal vascular parameters for cynomolgus macaques using FFA and OCT-A to help establish an optical parameter database for cynomolgus macaques and compare VD between healthy humans and cynomolgus macaques to promote choroid-retinopathy research. Trial registration: Current Controlled Trials and surrounding using OCT-A. Four levels of choroid-retinal capillary networks are present in the macula of cynomolgus macaques, including the superficial layer, the deep retinal layer, the outer retinal layer, and the choroid capillary layer. A comparison of the complete en-face VD among the four layers revealed the highest VD in the choroid capillary layer. are consistent with Florence Coscas ’ research in humans1. the deep layer formed a homogenous capillary vortex24, superficial layer formed capillaries VD


3
The retina plays a vital role in vision. The metabolic activity of the retina is higher than other human tissues1,2; therefore, the vascular circulation of the retina is complex. A variety of techniques have been used to measure retinal perfusion. Fundus fluorescence angiography (FFA) is routinely used to evaluate retinal vascular retinopathy3 as it can analyse the choroid-retinal vasculature and show vascular leakage and neovascularization4. However, this method has limitations, such as the intravenous injection of contrast agents that can lead to some side effects5,6. Additionally, further details of the deeper capillary plexus are unable to be extracted from FFA images due to limited depth perception. In contrast, optical coherence tomography angiography (OCT-A) is a novel imaging method7-9that detects the flow of blood via intrinsic signals without requiring an intravenous agent. OCT-A visualizes and divides the retina into various layers, clearly showing the capillary vessels in each layer. The ability of OCT-A to quantify the blood flow and vascular density of the retinal choroidal circulation is essential to the study of ophthalmology worldwide.
Due to their close similarity to humans in terms of optical structure and function, nonhuman primates such as cynomolgus macaques play a crucial role in ophthalmic disease research10-13. The FFA and OCT-A vascular parameters for normal humans have been previously reported1,14,15, and researchers have reported flow perfusion parameters for normal monkeys16. However, very little literature is available on the vascular density parameters for the macula and optic disc of ocularly normal cynomolgus macaques, and the comparison of VD between healthy humans and cynomolgus macaques using OCT-A has not been conducted. Our study aimed to evaluate macular vascular circulation parameters in normal cynomolgus macaques using OCT-A and FFA and to compare the advantages and disadvantages of the two angiography techniques.
Furthermore, for a better use of nonhuman primate models in studies of ophthalmic diseases, we compared the vascular parameters between healthy humans and cynomolgus macaques.

Animals
Data were collected from five normal adult male cynomolgus macaques with a mean age of 4.60±0.55 years and a mean weight of 4.44±0.88 kg. The animal production licence number was SCXK(YUE)2014-0027 (GuangZhou Blooming-Spring Biological Technology Development Co., Ltd., Guangzhou, China). Before starting the experiments, all five animals were determined to be ocularly normal and healthy. After these experiments, the animals continued to receive good care and will be used in subsequent studies.
Anaesthesia was maintained during examinations by intravenously injecting supplemental doses of ZoletilTM 50 (1/3 of the initial dose) as needed. Body temperature was maintained at 37°C using a water circulating heating pad. Pupils were fully dilated to approximately 9 mm in diameter with tropicamide phenylephrine eye drops (0.5%, Santen pharmaceutical Co.,Ltd, Japan). Sodium hyaluronate eye drops (0.3%, Santen pharmaceutical Co.,Ltd, Japan) 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.

Healthy humans
Healthy humans were recruited prospectively from the Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Guangdong between December 2017 and December 2018.
The macula of 28 healthy humans (14 males and 14 females) with a mean age of 25.11±6.21 years and the optic discs of 9 healthy humans (4 males and 5 females) with a mean age of 28.56±6.78 years were measured. The Ethics Committee of the Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Guangdong approved the research protocol, which followed the recommendations of the Declaration of Helsinki. Written informed consent was obtained from each patient before any examinations were performed. Each

Statistical analysis
The test images acquired using FFA and OCT-A were loaded into Photoshop CS6. The FFA images for the macular and optic disc areas were obtained and enlarged to match the details observed in the OCT-A images. IBM SPSS statistics version 19.0 (SPSS, Inc. Chicago, USA) was used to analyse the data. The measurements are presented as means standard deviations (SD). The comparisons of VD in different layers and different regions of each layer were analysed using the Bonferroni analysis. The level of significance adopted in the present study was P<0.05.

Stages of FFA
Human FFA is mainly divided into the following stages: the early arterial stage, arterial stage, arteriovenous stage, venous stage and later stage. In our study, FFA images from cynomolgus macaques tended to exhibit similarities to human angiographic stages. As shown in Fig. 1, the mean arm retina circulation time (RCT) was 5''11±1''35 s, and most fluorescence dissipation occurred at 17'18±1'21 min.

VD of the fovea and parafoveal region
The superficial capillary plexus is readily observed in fluorescein angiographic images of the central macular region; however, FFA is unable to image this region at a greater depth.  Table 1 and Fig. 6. In the superficial layer, there are no significant differences between two groups. However, it is observed that VD in the healthy human retina is higher than in the cynomolgus macaque retina in the fovea area of deep layer.
VD of the optic disc and peripapillary region Compared to OCT-A, the fluorescein angiographic image of the optic disc and surrounding regions showed the RPC network less clearly, as shown in Fig. 3 and 5. In Table 2, a comparison of the measurements of two layers of the cynomolgus macaque retina revealed that the mean VDs of the entire en-face, ID region and peripapillary region were different (P<0.01). The VD of the ID region was higher in the nerve head. In the peripapillary region, the VD was much higher in the RPC layer (P<0.01). Regarding the six peripapillary regions of each retinal layer, a significantly higher VD was in the IT section than in the N section (P<0.05) of the nerve head layer. In the RPC layer, the VD was lower in the N quadrant than in the IT and ST quadrants (P<0.05). Fig. 6 and Table 2  The VD of the entire en-face and ID section was higher in the nerve head layer. In contrast, the VD of the peripapillary region was much higher in the RPC layer. In both optic disc layers, the VD in the N quadrant was lower than the IT quadrant. Overall, OCT-A provides better images of the RPC network. The blood supply associated with early optic disc lesions in patients with glaucoma is derived from the microcirculation of the posterior ciliary artery; hence, an observation of the RPC layer is beneficial in the early diagnosis of glaucoma25.
Despite the novelty of OCT-A, particularly in assessing different diseases26-30, researchers have not clearly determined how OCT-A may be used for disease management, particularly in animal models. Currently, nonhuman primates, particularly cynomolgus macaques, play a very important role in the research of ophthalmic diseases due to their similarities to humans in terms of the optical structure and function8-11. Therefore, a better understanding of traditional FFA and novel OCT-A parameters and a comparison of differences between healthy humans and cynomolgus macaques will help establish these techniques, particularly OCT-A, as methods for the diagnosis of diseases in animal models.
We further compared the VD of normal humans and cynomolgus macaques in the area of the nerve head, RPC network and the layers of the superficial and deep retina, and all of the data showed some similarities and differences between two groups. In macula, the VDs of the superficial and deep layer are similar in both cynomolgus macaques and healthy humans. Only in the fovea area of deep layer, VD of healthy human is much higher.
Significant differences in the whole RPC layer of the optic disc were observed between the two groups and in the nerve head layer, healthy humans present higher VDs in various sections. Differences between two groups of VD are more obvious in the optic discs than the macula. These meaningful similarities and differences should take into account in the animal researches about human optical diseases, especially vascular diseases in macula and optic discs.

Conclusions
In conclusion, we evaluated macular vascular circulation parameters in normal cynomolgus macaques using OCT-A and FFA and compared the advantages and disadvantages of the two angiography techniques. Furthermore, we analysed the VD parameters of the choroidretinal vasculature and compared the vascular parameters between healthy humans and cynomolgus macaques. Obviously, the retinal structure of cynomolgus macaques was very similar to healthy humans; thus, we can use this animal model to better study the development of human optical diseases. However, some differences in the VD were observed between the two groups, indicating that when we use animal models to study optical diseases, we should also consider the functional differences between animals and humans and take these into account in the animal researches about human optical diseases, especially vascular diseases in macula and optic discs.. Overall, our research provides the normal vascular parameters of cynomolgus macaques and healthy humans, promoting the establishment of an eye parameter database for nonhuman primates and animal models of ophthalmic diseases. However, our study has certain limitations that must be addressed.
For example, we used cynomolgus macaques as the experimental animal and we did not clearly determine whether these results are applicable to other species. Our future goal is to conduct an extensive study across different species, comparing the similarities and differences in FFA and OCT-A parameters between healthy humans and nonhuman species to facilitate the research of ophthalmic diseases.         Histogram of vessel density (VD) in macula and optic disc of cynomolgus macaques and healthy humans.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.