1. Fundus photography
The cynomolgus monkey retina (Fig. 1A) appeared similar to the human retina on fundus photographs, with a clear optic nerve head, macula, and retinal vessels. The optic nerve head was oval (vertical in the long axis) and orange − red. Its boundary was clear and the cup to disc (C/D) ratio was about 0.2. Retinal arteries were bright red, veins were dark red, and the mean ratio of artery to vein diameter (A/V) was about 2:3. The macula was located on the temporal side of the optic disc, 1.5 vertical optic disc diameters (1.5 PD) from the optic disc. It was taupe in color and free of vessels. The healthy retina was flat and even, pigmentation was symmetrical in all directions.
Figure 1B presents FPs of New Zealand rabbit retina showing the optic nerve head, retinal vessels, and choroid. The optic nerve head was oval (horizontal in the long axis) and orange − red. The boundary was unclear and the C/D ratio was about 0.4 − 0.5. Ivory-colored myelinated nerve fibers and retinal vessels were observed passing through the optic disc. Compared to retinal veins, retinal arteries were brighter and thinner (A/V of about 2:3). Healthy New Zealand rabbit retinas were flat and even, no macular-like structure and pigment were observed. The choroid was visible beneath the retina but the macula could not be distinguished.
Fundus photographs of SD rats revealed a clear optic nerve head, retinal vessels, choroid (Fig. 1C). The optic nerve head was round and light yellow. The boundary was clear but with no obvious optic cup structure. No macula was detected, too. Retinal vessels were radially distributed. Healthy retinas were flat and even, without pigmentation. Circuitous choroid vessels were observed beneath the retina.
The BALB/c mouse retina (Fig. 1D) appeared similar to the SD rat retina on fundus photographs. The optic nerve head was quasi-circular, light yellow, and exhibited no unclear boundary or optic cup structure. No macula was observed. Retinal vessels were radially distributed. Healthy retinas of BALB/c mice were flat and even, with no pigment. Choroid was observed beneath the retina but the fine structure was unclear. New Zealand rabbits, SD rats, and BLAB/c mice are albino strains. In New Zealand rabbits and SD rats, the individual structures and distribution of choroid vessels were clearly visible beneath the retina. However, the choroid of BLAB/c mice was indistinct.
2. Fundus Fluorescence Angiography
After passing through the optic disc, the two retinal arteries of cynomolgus monkey bifurcated to form 4 arteries supplying the superior temporal, inferior temporal, superior nasal, and inferior nasal quadrants of the retina. The retinal veins ran in parallel with the arteries and converged first into upper and lower veins then a central vein before leaving the retina. The mean time from injection of fluorescein sodium into the monkey small saphenous vein to appearance in the retina was 10.7 ± 0.6 s. During the early phase of angiography (0 − 2 min post-injection), individual choroid capillaries filled with fluorescent dye and progressively became indistinguishable. Then the primary and secondary arteries filled, followed by the secondary and primary veins. The optic disc was also stained. During the late phase (8 − 10 min), fluorescence in retinal vessels faded and the optic disc appeared as a halo due to residual staining. There were no vessels at the macula, so this area remained fluorescence-free throughout image acquisition (Fig. 2A).
The two primary retinal arteries of New Zealand rabbits distributed to the nasal and temporal sides of the optic disc, emitting smaller branches along the way. Two large veins ran in parallel with the arteries, then passed through the optic disc. Circulation time from injection of fluorescein sodium into the ear vein to the retina was 8.4 ± 2.1 s. During the early phase, choroid became visible, followed by retinal arteries and veins in succession. During the late phase (6 − 10 min post-injection), vascular fluorescence faded, and optic disc fluorescence eventually disappeared. Individual choroid vessels filled with fluorescent tracer during the early phase and eventually became indistinguishable. The fluorescence faded more rapidly from choroid than from retinal vessels (Fig. 2B).
SD rat retinal vessels emerged from the optic disc, travelling straight and then radially. An average of 13.6 ± 2.3 vessels was observed in each rat retina. During angiography, retinal vessels became fluorescent rapidly and faded quickly. Due to the absence of pigment, the background fluorescence from the choroid was strong and interfered with the resolution of retinal angiography. However, vessels of the choroid may have filled and emptied rapidly compared to the acquisition rate, such that much of this process was missed (Fig. 3A).
Retinal vessels of BLAB/c mice emerged from the optic disc and formed an average of 10.7 ± 1.3 branches that distributed radially. During the early phase of FFA, retinal vessels gradually filled with fluorescein, and individual capillaries could be distinguished. During the late phase, large branches emptied but the vascular walls remained stained. Like SD rats, background fluorescence from the choroid was strong (Fig. 3B).
3. Optical Coherence Tomography
The structure of the cynomolgus monkey retina resembled that of the human retina on OCT images. These images distinguished 11 retinal layers, the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), external limiting membrane (ELM), myoid zone, ellipsoid zone, photoreceptor outer segment (OS), retinal pigment epithelium and Bruch`s complex (RPE/BM) [8–10], in addition to the underlying choroid (Fig. 4A). Macular foveal thickness did not differ between right eyes and left eyes (207.17 ± 14.77 µm vs. 209.43 ± 5.26 µm, p > 0.05). Alternatively, RNFL thickness differed significantly among the 4 quadrants (Table 1) (p = 0.001), with significant pair-wise differences between temporal and inferior (p = 0.008), superior and nasal (p = 0.005), and inferior and nasal (p = 0.001) quadrants.
Outside the optic disc, 11 layers were still observed in New Zealand rabbits` retina, including (RNFL, GCL, IPL, INL, OPL, ONL, incomplete ELM, photoreceptor inner segment (IS), junction of outer and inner segment, photoreceptor outer segment, and RPE/BM) (Fig. 4B) [11–13]. The average thickness of New Zealand rabbit retina was 251.20 ± 34.78 µm. Retinal thickness values within a 3 − 6 mm annular region centered on the optic disc are presented in Table 1. There was no significant difference between temporal and nasal retina thickness (p > 0.05). Due to the large optic disc of New Zealand rabbits, the annular region within 1–3 mm and RNFL thickness could not be measured.
Due to the multitude of retinal vessels in SD rats, many vascular shadows were observed by OCT, which partly obscured the RNFL. Nonetheless, 9 retinal layers could still be distinguished (RNFL, GCL, IPL, INL, OPL, ONL, ELM, IS/OS, and RPE/BM) (Fig. 4C) [14, 15]. Retina and RNFL thickness values across the retina are summarized in Table 1. Statistically significant differences in rat retinal thickness were found between annular regions of diameter 1 − 3 mm and 3 − 6 mm (p = 0.000), and between superior and inferior quadrants within the annular region of diameter 1 − 3 mm (p = 0.01). Mean RNFL thickness also differed significantly among the 4 quadrants (p = 0.000) and the inferior quadrant differed significantly from all three other quadrants (temporal p = 0.000, superior p = 0.000, nasal p = 0.022).
Like SD rats, numerous vascular shadows were seen on OCT images of BALB/c mouse retina. Nonetheless, 9 layers could be resolved (RNFL, GCL, IPL, INL, OPL, ONL, ELM, IS/OS, and RPE/BM) (Fig. 4D) [16]. The thickness distribution in different areas of retina and RNFL are summarized in Table 1. There was a statistically significant difference in retinal thickness between the annular region 1 − 2.22 mm from the optic disk and that 2.22 − 3.45 mm from the optic disk (p = 0.000), but no significant differences among quadrants within these annular regions (p > 0.05). Mean RNFL thickness did not differ among quadrants (p > 0.05).
Table 1
Thickness Distribution of Retina and RNFL (µm)
| Area | Temporal | Superior | Nasal | Inferior | Mean ± SD |
Monkey | RNFL | 82.93 ± 4.62 | 124.51 ± 20.49 | 56.07 ± 4.62 | 145.56 ± 22.98 | 113.19 ± 16.27 |
Rabbit | Retina | 3-6mm* | 304.6 ± 65.69 | 163.2 ± 30.04 | 308.1 ± 35.18 | 228.9 ± 44.04 | 251.20 ± 34.78 |
Rat | Retina | 1-3mm* | 242.11 ± 11.55 | 234.22 ± 11.97 | 245.94 ± 11.12 | 258.89 ± 21.73 | 245.29 ± 12.19 |
| 3-6mm* | 229.72 ± 8.96 | 224.94 ± 8.93 | 226.89 ± 7.73 | 234.44 ± 15.27 | 229.00 ± 9.46 |
RNFL | 45.47 ± 2.45 | 43.56 ± 2.66 | 47.47 ± 2.45 | 51.98 ± 4.00 | 47.33 ± 2.58 |
Mouse | Retina | 1-2.22mm* | 215.47 ± 12.82 | 218.47 ± 13.46 | 214.44 ± 16.72 | 214.22 ± 11.86 | 215.65 ± 12.61 |
| 2.22-3.45mm* | 227.22 ± 8.71 | 233.33 ± 19.88 | 229.92 ± 17.89 | 234.47 ± 17.68 | 231.24 ± 13.74 |
RNFL | 39.18 ± 8.08 | 37.56 ± 7.06 | 39.76 ± 7.65 | 37.91 ± 8.00 | 38.32 ± 7.34 |
*1-3mm: in an annular region centered on the optic disc with a diameter of 1–3 mm 3-6mm: in an annular region centered on the optic disc with a diameter of 3–6 mm 1-2.22mm: in an annular region centered on the optic disc with a diameter of 1-2.22 mm 2.22-3.45mm: in an annular region centered on the optic disc with a diameter of 2.22–3.45 mm RNFL: retinal nerve fiber layer |