
==== Front
Invest Ophthalmol Vis Sci
Invest Ophthalmol Vis Sci
IOVS
Investigative Ophthalmology & Visual Science
0146-0404
1552-5783
The Association for Research in Vision and Ophthalmology

39250121
10.1167/iovs.65.11.14
IOVS-24-40375
Retina
Retina
Peripapillary Retinal Nerve Fiber Layer and Macular Ganglion Cell-Inner Plexiform Layer Parameters and Their Associated Factors in Cynomolgus Macaques
pRNFL/mGCL/mIPL in Cynomolgus Macaques
Zhang Yuan 1
Ye Guitong 1
Chen Kezhe 1
Huang Shaofen 1
Xie Rui 1
Chen Jianqi 1
Liu Wei 2
Wang Zhiwei 2
Luo Ruiyu 1
Zhan Jinan 1
Zhuo Yehong 1
Li Yiqing 1
Zhu Yingting 1
1 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China
2 Huazhen Biosciences, Guangzhou, China
# Correspondence: Yehong Zhuo, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yatsen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, 7 Jinsui Road, Tianhe District, Guangzhou 510623, China; zhuoyh@mail.sysu.edu.cn.
Yiqing Li, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yatsen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, 7 Jinsui Road, Tianhe District, Guangzhou 510623, China; liyiqing3@mail.sysu.edu.cn.
Yingting Zhu, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yatsen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, 7 Jinsui Road, Tianhe District, Guangzhou 510623, China; zhuyt35@mail.sysu.edu.cn.
* YZ and GY contributed equally to this study and should be considered as co-first authors.

09 9 2024
9 2024
65 11 1409 8 2024
09 5 2024
Copyright 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose

The purpose of this study was to define the normal range of peripapillary retinal nerve fiber layer (pRNFL), macular ganglion cell layer (mGCL), and macular inner plexiform layer (mIPL) thickness in cynomolgus macaques, and explore their inter-relationship and correlation with age, refractive errors, and axial length (AL).

Methods

In this cross-sectional study, we measured biometric and refractive parameters, and pRNFL/mGCL/mIPL thickness in 357 healthy cynomolgus macaques. Monkeys were divided into groups by age and spherical equivalent (SE). Correlation and regression analyses were used to explore the relationship between pRNFL and mGCL/mIPL thickness, and their correlation with the above parameters.

Results

The mean age, SE, and AL were 14.46 ± 6.70 years, −0.96 ± 3.23 diopters (D), and 18.39 ± 1.02 mm, respectively. The mean global pRNFL thickness was 95.06 ± 9.42 µm (range = 54–116 µm), with highest values in the inferior quadrant, followed by the superior, temporal, and nasal quadrants (P < 0.001). Temporal pRNFL thickness correlated positively with age (r = 0.218, P < 0.001) and AL (r = 0.364, P < 0.001), and negatively with SE (r = −0.270, P < 0.001). In other quadrants, pRNFL thickness correlated negatively with age and AL, but positively with SE. In the multivariable linear regression model, adjusted for sex and AL, age (β = −0.350, P < 0.001), and SE (β = 0.206, P < 0.001) showed significant associations with global pRNFL thickness. After adjusting for age, sex, SE, and AL, pRNFL thickness positively correlated with mGCL (β = 0.433, P < 0.001) and mIPL thickness (β = 0.465, P < 0.001).

Conclusions

The pRNFL/mGCL/mIPL thickness distribution and relationship with age, AL, and SE in cynomolgus macaques were highly comparable to those in humans, suggesting that cynomolgus monkeys are valuable animal models in ophthalmic research.

cynomolgus macaques
retinal nerve fiber layer (RNFL) thickness
refractive errors
axial length (AL)
==== Body
pmcMyopia affects nearly 34% of the global population, with an anticipated rise to 50% by 2050.1 Severe (high) myopia during childhood is associated with increased risk for ophthalmic complications that can ultimately lead to vision loss.2 Additionally, myopic enlargement of the globe during childhood, leading to axial elongation, may be accompanied by changes in many structures, including the optic nerve head (ONH), retina, choroid, and sclera.3–5

Peripapillary retinal nerve fiber layer (pRNFL) and optic nerve structural parameters are important indicators in the application of optical coherence tomography (OCT) for glaucoma diagnosis.6,7 However, in patients with high myopia, ONH size and structure can change, such as with peripapillary atrophy. Consequently, pRNFL thickness measurements in these patients are often inaccurate.8–11 Complicating the interpretation of OCT results and thus necessitating careful consideration in clinical evaluations. Accordingly, it is essential to investigate the relationship between the pRNFL thickness and spherical equivalent (SE) and axial length (AL). Furthermore, studying pRNFL thickness may improve our understanding of the pathological mechanisms of myopia, although available data on the effect of myopia on pRNFL thickness are conflicting.

Additionally, the reliability of pRNFL measurements is significantly affected by cup-to-disc ratio variability and ONH vascular structure changes and tilt angle differences.12,13 Compared with the ONH, the macular region has a relatively stable structure, with more than 50% of retinal ganglion cells concentrated in the macula and distributed in multiple layers.14 Some studies have suggested that macular ganglion cell-inner plexiform layer (mGC-IPL) thickness has comparable or better glaucoma detection capability than pRNFL thickness in patients with high myopia.15,16

Nonhuman primates have been recognized as superior models for visual and neuroscience research.17 Unlike other animal models commonly used in myopia studies, such as chickens, mice, guinea pigs, and tree shrews,18,19 cynomolgus macaques have more human-resembling eye structures, particularly the macula structure and vitreous volumes. Nonetheless, few studies have described normative pRNFL, macular ganglion cell layer (mGCL), mIPL thickness values, and distribution in healthy cynomolgus macaques.

The aim of this cross-sectional study is to provide normative data for pRNFL, mGCL, and mIPL thickness in 357 healthy cynomolgus macaques and to determine their relationships with age, sex, AL, and refractive error. Defining reference ranges for ophthalmologic parameters and their inter-relations will provide a basis for using cynomolgus macaques as experimental models.

Methods

Animals

This research strictly adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Huazhen Biosciences (HZ-EXF-001) and Zhongshan Ophthalmic Centre (W202203). The cynomolgus monkeys (total 395) used in this study were housed at Huazhen Biosciences in air-conditioned rooms, with temperatures maintained at 16°C to 26°C, relative humidity at 40% to 70%, and natural diurnal rhythms. None of these monkeys had ever been subjected to any ocular manipulation. Monkeys with a history of trauma, severe cataracts, and fundus lesions were excluded.

According to the time of reproductive debut and sexual maturity, cynomolgus macaques go through infant (0–1.5 years), juvenile (1.5–4 years), subadult (4–9 years), adult (9–20 years), and geriatric (20–25 years) age stages.20 Accordingly, our cynomolgus population was classified into four age groups: juvenile, subadult, adult, and geriatric.

Ophthalmological Examination

Animal Preparation

Following intramuscular anesthesia with Zoletil 50 at a dose of 10 mg/kg, the monkeys were placed in the prone position on a flat but comfortable countertop. An experienced technician secured the monkey's head in the primary gaze position and lifted the eyelid to ensure full corneal exposure. An eyelid speculum was deployed to aid in the measurement process. Prior to the examination, cycloplegia was induced by administering 3 drops of 1% tropicamide; the first 2 drops 5 minutes apart, and the third after 20 minutes.

In addition to the below, other examinations were conducted for ocular diseases screening (e.g. fundus photography) according to standard operating procedures.

Autorefraction Measurement

Autorefraction measurement was conducted by an experimental optometrist using Fario FXR-710 (Fario, Zhejiang, China), which integrates optometry and keratometry functions. Measurement was commenced when the pupil diameter expanded to a minimum of 6 mm, typically occurring 15 to 30 minutes after the administration of the third tropicamide drop. Careful adjustments were made to ensure the monkey's eyes were precisely aligned with the instrument's lens. The device automatically located the center of the pupil and proceeded with the measurement.

Metrics included spherical refraction (SR), cylinder refraction (CR), and corneal radius of curvature (CRC), which were recorded at least three times. The SE was calculated automatically. Data were considered reliable and retained for analysis when the error margins for SR or CR were less than 0.25 diopters (D). Subsequently, the mean SR, CR, and SE were calculated.

For analysis purposes, monkeys were divided into four refraction groups according to the mean SE value: hyperopia (SE > +0.50 D), emmetropia (−0.50 D ≤ SE ≤ +0.50 D), mild/moderate myopia (−0.50 D > SE > −6.00 D), and high myopia (SE ≤ −6.00 D).

OCT Measurement

OCT was performed using the Heidelberg Spectralis HRA OCT (Heidelberg Engineering, Heidelberg, Germany). Scans centered on the optic disc were used for peripapillary measurements and scans centered on the fovea for macular measurements (Fig. 1). All scans were obtained using the high-resolution mode.

Figure 1. Spectral-domain optical coherence tomography images of the right eye of a healthy cynomolgus macaque. (A) A 25-line horizontal raster scan centered on the macula (20 degrees × 20 degrees, 6.0 mm × 6.0 mm). The average thickness of macular ganglion cell layer (mGCL) and macular inner plexiform layer (mIPL) was measured in nine different regions based on the ETDRS sectors. (B) High-resolution mode scan centered on the optic disc to measure peripapillary retinal nerve fiber layer (pRNFL) thickness. (C, D) Measurement reports for mGCL/mIPL, respectively. (E) Display of macular and optic disc scan areas. In the macular scan: C, central fovea (1 mm); inner ring, 1–3 mm from the central fovea; outer ring, 3–6 mm from the central fovea. S3, superior inner ring, 1–3 mm from the central fovea; T3, temporal inner ring; I3, inferior inner ring; N3, nasal inner ring; S6, superior outer ring, 3–6 mm from the central fovea; T6, temporal outer ring; I6, inferior outer ring; N6, nasal outer ring. (F) Measurement report for pRNFL thickness. G, global; ILM, internal limiting membrane; INF, inferior; N, nasal; NAS, nasal; NI, nasal inferior; NS, nasal superior; RNFL, retinal nerve fiber layer; T, temporal; TI, temporal inferior; TMP, temporal; TS, temporal superior; SUP, superior.

pRNFL Measurements

The device's segmentation algorithms adeptly delineated the pRNFL. Manual adjustments were made when necessary to guarantee the highest level of accuracy. The Heidelberg OCT software automatically assessed pRNFL thickness across six distinct sectors (see Fig. 1): temporal (T; 315 degrees to 45 degrees), temporal-superior (TS; 45 degrees to 90 degrees), temporal-inferior (TI; 90 degrees to 135 degrees), nasal (N; 135 degrees to 225 degrees), nasal-superior (NS; 225 degrees to 270 degrees), and nasal-inferior (NI; 270 degrees to 315 degrees). The mean global pRNFL thickness was calculated by averaging the mean pRNFL thickness values of the 360 degrees measurements.

mGCL and mIPL Measurements

A 25-line horizontal raster scan (20 degrees × 20 degrees, 6.0 × 6.0 mm) centered on the fovea and 9 frames averaged in each OCT B-scan. The average mGCL and mIPL thickness was measured in nine separate areas (based on Early Treatment Diabetic Retinopathy Study [ETDRS] sectors) within the macula. For ease of analysis, the region within 1 mm of the foveal center was defined as C. The superior, inferior, nasal, and temporal regions within the “inner ring” (1–3 mm from the foveal center) were defined as S3, I3, N3, and T3, and those within the “outer ring” (3–6 mm from the foveal center) were defined as S6, I6, N6, and T6, respectively (see Fig. 1).

AL Measurement

The AL was measured as the distance between the anterior cornea and the retinal pigment epithelium using a non-contact biometer (Lenstar LS 900; Haag Streit AG, Koeniz, Switzerland). All measurements were conducted at least five times.

Statistical Analysis

Because there was a strong correlation (Pearson's r = 0.509–0.979) between the left and right eye measurements, only the right eye data were used for analysis, except for monkeys with right eye diseases, for whom the left eye data were used.

Normality of data distribution was evaluated using the Kolmogorov–Smirnov test. Quantitative data with normal distribution were summarized as mean values with standard deviations, those with skewed distribution as median values with interquartile ranges, and qualitative data as counts and percentages. Two-tailed independent sample t-tests were used to detect differences between sexes and 1-way analysis of variance to detect differences among age, and SE groups. Bonferroni correction was used to control for potential false discoveries in multiple comparisons.

Univariate analysis (Pearson's correlation coefficient) and multiple linear regression analysis were used to determine the relationship between pRNFL parameters (independent variable) and mGCL/mIPL parameters (dependent variable), as well as their respective relationships with sex, age, SE, and AL. The variance inflation factor was used to diagnose multicollinearity between age and pRNFL thickness, age and SE, and SE and AL, with all variance inflation factor values being less than 3.

All statistical analyses were performed using IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). P values less than 0.05 were considered to indicate statistical significance.

Results

Demographics

Of the 395 cynomolgus monkeys screened, 357 (115 male monkeys and 242 female monkeys) were included in the study. Among them, three monkeys had right eye diseases; for these monkeys, the left eye data were used. The majority of enrolled animals were female (n = 242, 68%). The mean age and weight were 14.46 ± 6.70 (2.96–26.15) years and 4.51 ± 1.16 (2.44–8.52) kg, respectively (Table 1).

Table 1. General Characteristics of the Study Population by Sex

Characteristic	Total	Range	Male (n, %)	Female (n, %)	P Value	
N, n (%)	357	/	115 (32)	242 (68)	/	
Age, y	14.46 ± 6.70	2.96 to 26.15	11.32 ± 7.07	15.96 ± 5.98	<0.001	
Age distribution						
1.5–4 y (juvenile)	33 (9%)	2.96 to 3.99	18 (55)	15 (45)	/	
4–9 y (subadult)	75 (21%)	4.06 to 8.98	41 (55)	34 (45)	/	
9–20 y (adult)	185 (52%)	10.38 to 19.48	35 (19)	150 (81)	/	
20–26 y (geriatric)	64 (18%)	19.50 to 26.15	21 (33)	43 (67)	/	
Weight, kg	4.51 ± 1.16	2.44 to 8.52	6.08 ± 1.06	4.15 ± 0.83	0.067	
Data are presented as mean ± standard deviation or n (%).

P values were obtained using the two-tailed independent sample t-tests. P < 0.05 was consider statistically significant.

Overall, the SE ranged from −23.21 to 8.5 D (mean = −0.96 ± 3.23 D), and AL ranged from 16.24 to 22.24 mm (mean = 18.39 ± 1.02 mm). Although the mean SE was similar in both sex groups, the mean AL was shorter in female monkeys than in male monkeys. The mean pRNFL thickness was similar in both sexes for all sectors except the nasal superior sector, for which mean pRNFL thickness was significantly greater in male monkeys than in female monkeys (100.61 ± 19.44 µm vs. 96.45 ± 16.25 µm, P = 0.005). The mGCL thickness in I3 and mIPL thickness in S3/T3/I3 were significantly greater in male monkeys than in female monkeys, whereas the mIPL thickness in I6/N6 was significantly greater in female monkeys than in male monkeys (all P < 0.05; Table 2).

Table 2. Distributions of Peripapillary Retinal Nerve Fibre Layer (pRNFL), Macular Ganglion Cell Layer (mGCL), Macular Inner Plexiform Layer (mIPL), Spherical Equivalent, and Axial Length by Sex

Parameters	Total	Range	Male (n = 115)	Female (n = 242)	P Value	
pRNFL, µm						
 S	115.30 ± 15.21	51 to 170	116.75 ± 15.37	114.66 ± 15.14	0.285	
 N	62.63 ± 13.96	20 to 107	65.51 ± 14.90	61.29 ± 1334	0.129	
 I	131.83 ± 15.89	64 to 175	132.07 ± 17.05	131.72 ± 15.39	0.075	
 T	70.56 ± 13.78	30 to 117	71.25 ± 14.70	70.27 ± 13.37	0.571	
 G	95.06 ± 9.42	54 to 116	96.45 ± 9.35	94.43 ± 9.42	0.463	
 NS	97.82 ± 17.40	36 to 151	100.61 ± 19.44	96.45 ± 16.25	0.005 *	
 TS	132.42 ± 18.22	70 to 194	132.88 ± 18.48	132.33 ± 18.06	0.906	
 NI	144.38 ± 19.36	65 to 195	145.49 ± 18.68	143.84 ± 19.73	0.684	
 TI	119.30 ± 24.35	40 to 201	118.68 ± 26.82	119.63 ± 23.14	0.039	
mGCL, µm						
 Whole	42.14 ± 3.20	33 to 51	42.35 ± 3.03	42.03 ± 3.29	0.324	
 Inner ring average	43.74 ± 4.11	32 to 55	44.63 ± 3.89	43.28 ± 4.16	0.109	
 Outer ring average	36.78 ± 3.01	29 to 45	36.51 ± 2.79	36.90 ± 3.11	0.074	
 S3	45.50 ± 4.42	31 to 37	46.02 ± 4.48	45.20 ± 4.65	0.500	
 T3	40.36 ± 4.53	27 to 52	41.68 ± 4.37	39.62 ± 4.80	0.158	
 I3	44.63 ± 4.95	23 to 57	45.58 ± 4.32	44.08 ± 5.56	0.021	
 N3	44.32 ± 4.63	32 to 56	45.25 ± 4.58	43.80 ± 4.75	0.247	
 S6	34.88 ± 3.15	25 to 42	34.58 ± 2.95	34.91 ± 3.54	0.121	
 T6	34.84 ± 3.44	26 to 44	34.52 ± 3.50	34.90 ± 3.55	0.812	
 I6	36.52 ± 3.90	21 to 46	36.32 ± 3.14	36.59 ± 4.40	0.008	
 N6	40.90 ± 3.28	32 to 49	40.68 ± 2.94	40.97 ± 3.68	0.063	
mIPL, µm						
 Whole	34.27 ± 2.35	27 to 41	34.48 ± 2.19	34.18 ± 2.43	0.082	
 Inner ring average	39.13 ± 3.04	29 to 48	39.65 ± 2.75	38.87 ± 3.16	0.018	
 Outer ring average	29.42 ± 2.29	24 to 38	29.31 ± 2.11	29.48 ± 2.38	0.099	
 S3	39.65 ± 3.45	28 to 49	40.11 ± 3.20	39.13 ± 4.39	0.034	
 T3	38.48 ± 3.16	30 to 48	39.10 ± 2.90	38.13 ± 3.34	0.040	
 I3	38.02 ± 3.62	24 to 47	38.57 ± 2.98	37.63 ± 4.19	0.001	
 N3	40.29 ± 3.54	28 to 50	40.94 ± 3.28	39.95 ± 3.85	0.177	
 S6	27.62 ± 2.54	19 to 33	27.57 ± 2.37	27.56 ± 2.80	0.129	
 T6	29.91 ± 2.48	24 to 38	29.91 ± 2.59	29.92 ± 2.47	0.531	
 I6	28.80 ± 2.85	19 to 38	28.64 ± 2.43	28.88 ± 3.15	0.032	
 N6	31.32 ± 2.84	24 to 41	31.19 ± 2.48	31.40 ± 3.08	0.016	
SE, D	−0.96 ± 3.23	8.50 to −23.21	−1.12 ± 3.64	−0.93 ± 2.97	0.476	
AL, mm	18.39 ± 1.02	16.24 to 22.24	18.52 ± 1.11	18.32 ± 0.91	0.027 *	
AL, axial length; G, global; I, inferior; I3, inferior inner ring; I6, inferior outer ring; N, nasal; N3, nasal inner ring; N6, nasal outer ring; NI, nasal inferior; NS, nasal superior; S, superior; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; SE, spherical equivalent; T, temporal; T3, temporal inner ring; T6, temporal outer ring; TI, temporal inferior; TS, temporal superior.

Data are presented as mean ± standard deviation.

The P values were calculated using two-tailed independent sample t-tests, refers to the comparison result between male monkeys and female monkeys.

* P < 0.05 indicates statistical significance (bold font).

Distribution of pRNFL/mGCL/mIPL Thickness

The mean global pRNFL thickness was 95.06 ± 9.42 (54–116) µm. The pRNFL was thickest inferiorly (131.83 ± 15.89 µm) and superiorly (115.30 ± 15.21 µm), thinner temporally (70.56 ± 13.78 µm), and thinnest nasally (62.63 ± 13.96 µm, P < 0.05). The mGCL/mIPL thickness is very small at the foveal center, these values were omitted from the analysis. Overall, the average thickness of the mGCL and mIPL was significantly greater in the nasal than in the temporal quadrant, and in the inner than in the outer ring (all P < 0.05; see Table 2, Fig. 2).

Figure 2. Distribution of macular ganglion cell layer (mGCL), macular inner plexiform layer (mIPL) and peripapillary retinal nerve fiber layer (pRNFL) thickness in different retinal sectors and areas. (A) Line graph of pRNFL. (B) Box and whiskers graph of pRNFL thickness. G, global; N, nasal; NI, nasal inferior; NS, nasal superior; T, temporal; TI, temporal inferior; TS, temporal superior. (C) Box and whiskers graph of mGCL/mIPL/mGCL+mIPL thickness. C, central fovea (1 mm); I3, inferior inner ring; I6, inferior outer ring; N3, nasal inner ring; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; T3, temporal inner ring; T6, temporal outer ring.

Figure 3. Box plots of macular ganglion cell layer (mGCL), macular inner plexiform layer (mIPL) and peripapillary retinal nerve fiber layer (pRNFL) thickness by age groups and spherical equivalent groups. G, global; I, the inner ring, 1–3 mm from the central fovea; I3, inferior inner ring; I6, inferior outer ring; N, nasal; N3, nasal inner ring; N6, nasal outer ring; NI, nasal inferior; NS, nasal superior; O, the outer ring; 3–6 mm from the central fovea; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; T, temporal; T3, temporal inner ring; T6, temporal outer ring; TI, temporal inferior; TS, temporal superior; W, the whole ring (except central fovea [1 mm]).

Figure 4. Scatterplots showing the distribution of macular ganglion cell layer (mGCL), macular inner plexiform layer (mIPL) in different areas.

Distribution of pRNFL/mGCL/mIPL Thickness by Age

In the temporal quadrant, pRNFL thickness was greater in the adult group (73.11 ± 14.05 µm) than in the subadult (66.45 ± 10.91 µm, P = 0.001) and juvenile groups (63.42 ± 11.37 µm, P = 0.001). In contrast, in other quadrants and sectors, the mean pRNFL thickness was greatest in the juvenile and subadult groups, and smallest in the geriatric group (P < 0.001). No significant differences were observed between the juvenile and subadult groups. For mGCL/mIPL thickness, significant differences between age groups were observed only in the “outer ring,” primarily between the subadult and adult/geriatric groups (Table 3, Fig. 3).

Table 3. Distributions of Peripapillary Retinal Nerve Fiber Layer (pRNFL), Macular Ganglion Cell Layer (mGCL), Macular Inner Plexiform Layer (mIPL), Spherical Equivalent, and Axial Length by Age and Correlation With Age

Parameters	Juvenile (n = 31)	Subadult (n = 75)	Adult (n = 185)	Geriatric (n = 66)	P Value	R	P Value	
pRNFL, µm								
 S	(98–141)121.39 ± 11.85	(100–153)123.16 ± 12.08	(59–170)112.80 ± 15.60	(78–152)110.69 ± 14.73	P < 0.001, Pa = 0.016, Pb = 0.012, Pc < 0.001, Pd < 0.001	−0.300	<0.001	
 N	(55–104)75.03 ± 12.07	(49–98)74.58 ± 10.61	(20–86)57.11 ± 11.07	(32–107)58.61 ± 13.41	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001, Pd < 0.001	−0.545	<0.001	
 I	(121–161)139.16 ± 10.38	(94–168)140.64 ± 14.83	(84–175)128.93 ± 15.50	(86–165)126.19 ± 15.16	P < 0.001, Pa = 0.003, Pb = 0.001, Pc < 0.001, Pd < 0.001	−0.343	<0.001	
 T	(47–94)63.42 ± 11.37	(45–98)66.45 ± 10.91	(30–117)73.11 ± 14.05	(47–117)71.19 ± 15.01	P < 0.001, Pa = 0.001, Pc = 0.001	0.218	<0.001	
 G	(85–112)99.74 ± 6.54	(84–116)101.25 ± 7.78	(54–114)92.92 ± 9.16	(73–116)91.72 ± 8.95	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001, Pd < 0.001	−0.405	<0.001	
 NS	(105–167)137.26 ± 14.02	(95–192)140.82 ± 17.21	(70–194)130.11 ± 18.72	(99–174)127.39 ± 16.20	P < 0.001, Pc < 0.001, Pd < 0.001	−0.279	<0.001	
 TS	(76–141)105.65 ± 17.98	(72–151)105.52 ± 15.20	(44–151)94.80 ± 16.84	(58–138)93.96 ± 17.35	P < 0.001, Pa = 0.005, Pb = 0.011, Pc < 0.001, Pd = 0.001	−0.291	<0.001	
 TI	(112–181)153.10 ± 15.65	(103–195)154.79 ± 17.92	(65–192)140.62 ± 18.86	(98–180)138.93 ± 18.18	P < 0.001, Pa = 0.003, Pb = 0.004, Pc < 0.001, Pd < 0.001	−0.327	<0.001	
 NI	(95–157)125.32 ± 17.76	(66–201)126.41 ± 22.28	(59–195)117.20 ± 25.93	(52–172)113.57 ± 22.66	P = 0.011, Pd = 0.045	−0.177	0.001	
mGCL, µm								
 Whole	(35–47)40.50 ± 2.72	(35–51)42.92 ± 3.00	(35–51)42.36 ± 3.28	(33–48)41.37 ± 3.10	P = 0.001, Pe = 0.003, Pa = 0.019, Pd = 0.037	−0.046	0.419	
 Inner ring average	(34–54)43.68 ± 4.21	(34–55)44.08 ± 4.10	(34–54)43.95 ± 4.09	(32–49)42.61 ± 4.06	P = 0.221	−0.078	0.161	
 Outer ring average	(32–42)37.23 ± 2.72	(31–44)37.87 ± 2.60	(29–44)36.45 ± 3.06	(30–45)35.97 ± 3.16	P = 0.001, Pc = 0.004, Pd = 0.003	−0.222	<0.001	
 S3	(36–57)44.70 ± 4.71	(31–55)45.66 ± 4.77	(34–56)45.77 ± 4.30	(34–52)44.87 ± 4.13	P = 0.546	−0.014	0.806	
 T3	(28–49)40.73 ± 4.94	(31–52)40.35 ± 4.32	(27–50)40.23 ± 4.25	(27–51)39.13 ± 5.13	P = 0.066	−0.153	0.006	
 I3	(35–54)45.30 ± 3.78	(31–57)44.89 ± 4.55	(23–56)44.84 ± 5.31	(30–52)43.17 ± 4.82	P = 0.166	−0.088	0.113	
 N3	(33–56)43.97 ± 4.64	(32–55)44.40 ± 4.82	(33–54)44.68 ± 4.51	(32–51)43.26 ± 4.61	P = 0.434	−0.036	0.515	
 S6	(30–42)35.90 ± 2.78	(29–42)35.64 ± 2.58	(25–42)34.58 ± 3.38	(25–40)34.11 ± 3.13	P = 0.009, Pc = 0.046	−0.212	<0.001	
 T6	(30–41)35.50 ± 3.07	(29–44)36.03 ± 3.06	(26–44)34.48 ± 3.42	(28–43)33.94 ± 3.78	P = 0.001, Pc = 0.005, Pd = 0.005	−0.218	<0.001	
 I6	(26–41)36.50 ± 3.45	(27–44)37.64 ± 3.22	(21–45)36.11 ± 4.32	(25–46)36.11 ± 3.53	P = 0.045, Pc = 0.032	−0.133	0.017	
 N6	(36–46)41.43 ± 2.61	(32–48)41.79 ± 2.88	(32–49)40.71 ± 3.46	(32–49)39.89 ± 3.36	P = 0.016, Pd = 0.020	−0.186	0.001	
mIPL, µm								
 Whole	(30–39)34.45 ± 1.95	(29–41)34.76 ± 2.15	(27–41)34.23 ± 2.52	(27–40)33.59 ± 2.20	P = 0.076	−0.154	0.006	
 Inner ring average	(32–45)39.03 ± 2.84	(33–48)39.37 ± 2.93	(29–48)39.20 ± 3.21	(30–44)38.55 ± 2.77	P = 0.562	−0.071	0.202	
 Outer ring average	(26–34)29.88 ± 2.00	(25–35)30.15 ± 2.06	(24–38)29.25 ± 2.38	(24–36)28.63 ± 2.18	P = 0.002, Pc = 0.026, Pd = 0.003	−0.230	0.000	
 S3	(33–44)39.40 ± 2.69	(32–48)40.07 ± 3.37	(28–49)39.59 ± 3.62	(28–48)39.28 ± 3.43	P = 0.487	−0.091	0.101	
 T3	(32–46)38.07 ± 3.26	(32–48)38.59 ± 2.96	(30–47)38.63 ± 3.29	(30–47)38.06 ± 2.98	P = 0.695	−0.032	0.570	
 I3	(30–44)38.37 ± 3.16	(28–47)38.33 ± 3.27	(24–47)38.05 ± 3.86	(28–46)37.20 ± 3.55	P = 0.340	−0.092	0.099	
 N3	(32–47)40.30 ± 3.14	(33–50)40.51 ± 3.48	(29–50)40.39 ± 3.64	(28–48)39.65 ± 3.56	P = 0.681	−0.068	0.219	
 S6	(25–33)28.37 ± 2.08	(23–32)28.25 ± 2.15	(19–33)27.38 ± 2.71	(21–32)27.00 ± 2.53	P = 0.009, Pc = 0.039	−0.211	<0.001	
 T6	(25–35)30.13 ± 2.36	(26–37)30.47 ± 2.27	(24–38)29.90 ± 2.52	(24–37)29.07 ± 2.53	P = 0.038, Pd = 0.026	−0.168	<0.001	
 I6	(21–32)28.70 ± 2.63	(22–35)29.66 ± 2.41	(19–38)28.58 ± 3.12	(21–36)28.26 ± 2.50	P = 0.033, Pc = 0.045	−0.147	<0.001	
 N6	(28–37)32.30 ± 2.22	(27–37)32.22 ± 2.43	(24–41)31.08 ± 3.01	(24–38)30.17 ± 2.72	P < 0.001, Pb = 0.007, Pc = 0.037, Pd < 0.001	−0.255	<0.001	
SE, D	(−3.00 to 2.63)0.20 ± 1.31	(−11.46 to 2.13)−0.61 ± 2.34	(−23.21 to 7.21)−1.38 ± 3.72	(−13.42 to 3.29)−0.91 ± 2.85	P = 0.031	−0.131	0.014	
AL, mm	(16.42–18.10)17.28 ± 0.52	(16.43–20.67)17.60 ± 0.80	(17.29–22.24)18.71 ± 0.84	(18.00–21.99)18.76 ± 0.85	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001, Pd < 0.001	0.557	<0.001	
Data are presented as (ranges) and mean ± standard deviation.

S, superior; N, nasal; I, inferior; T, temporal; G, global; NS, nasal superior; TS, temporal superior; NI, nasal inferior; TI, temporal inferior; S3, superior inner ring, 1–3 mm from the central fovea; T3, temporal inner ring; I3, inferior inner ring; N3, nasal inner ring; S6, superior outer ring, 3–6 mm from the central fovea; T6, temporal outer ring; I6, inferior outer ring; N6, nasal outer ring; SE, spherical equivalent; AL, axial length; r, two-tailed Pearson's bivariate correlation coefficient.

P values without superscripts were calculated using multigroup ANOVA; P < 0.05 was considered statistically significant (bold font).

a = P value between juvenile and adult.

b = P value between juvenile and geriatric.

c = P value between subadult and adult.

d = P value between subadult and geriatric.

e = P value between juvenile and subadult.

Distribution of pRNFL/mGCL/mIPL Thickness by SE

Compared with those in the other groups, the mean pRNFL thickness in the high myopia group was persistently greater in the temporal quadrant (P < 0.0001), and persistently smaller (P < 0.001) in other quadrants. In the TS and TI sectors, no pRNFL difference was observed among the SE groups. For mGCL/mIPL thickness, significant differences between SE groups were observed in the “inner ring,” primarily between the high myopia and other groups (Table 4, Fig. 3).

Table 4. Distributions of Peripapillary Retinal Nerve Fiber Layer (pRNFL), Macular Ganglion Cell Layer (mGCL), Macular Inner Plexiform Layer (mIPL), Age, and Axial Length by Spherical Equivalent and Correlation With Spherical Equivalent

Parameters	Hyperopia (n = 130)	Emmetropia (n = 81)	Mild/Moderate Myopia (n = 117)	High Myopia (n = 29)	P Value	r	P Value	
pRNFL, µm								
 S	114.77 ± 13.22	117.57 ± 15.43	115.77 ± 15.67	105.67 ± 14.93	P < 0.001, Pa = 0.001, Pb < 0.001, Pc = 0.001	0.217	<0.001	
 N	63.72 ± 13.47	65.16 ± 11.77	61.71 ± 13.25	50.05 ± 16.26	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001	0.286	<0.001	
 I	132.78 ± 14.72	133.05 ± 16.81	131.11 ± 15.44	121.86 ± 12.73	P < 0.001, Pa < 0.001, Pb < 0.001, Pc = 0.005	0.291	<0.001	
 T	68.73 ± 11.12	67.42 ± 11.00	72.05 ± 14.68	81.91 ± 19.91	P < 0.001, Pa < 0.001, Pb < 0.001, Pc = 0.001	−0.270	<0.001	
 G	95.02 ± 9.02	95.86 ± 10.31	95.05 ± 8.76	89.83 ± 7.38	P = 0.001, Pa = 0.001, Pb = 0.001, Pc = 0.004	0.257	<0.001	
 NS	97.90 ± 15.16	101.31 ± 17.45	97.42 ± 17.32	84.59 ± 18.10	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001	0.273	<0.001	
 TS	131.60 ± 16.50	133.80 ± 19.42	133.01 ± 18.33	126.94 ± 18.58	P = 0.141	0.121	0.024	
 TI	143.81 ± 20.07	144.31 ± 20.80	143.71 ± 18.51	143.98 ± 13.67	P = 0.859	0.062	0.245	
 NI	121.83 ± 22.98	121.79 ± 23.86	118.52 ± 24.32	99.73 ± 21.60	P < 0.001, Pa < 0.001, Pb < 0.001, Pc < 0.001	0.333	<0.001	
mGCL (µm)								
 Whole	42.40 ± 3.30	42.72 ± 3.21	41.87 ± 3.15	40.66 ± 2.73	P = 0.027, Pb = 0.032	0.189	0.001	
 Inner ring average	44.13 ± 4.23	44.39 ± 4.15	43.41 ± 4.05	41.71 ± 3.45	P = 0.027, Pb = 0.027	0.200	0.000	
 Outer ring average	36.95 ± 3.12	37.29 ± 2.74	36.51 ± 3.00	35.43 ± 3.04	P = 0.020, Pa = 0.050, Pb = 0.031	0.179	0.001	
 S3	45.72 ± 4.69	45.88 ± 4.64	45.50 ± 4.13	43.73 ± 3.93	P = 0.214	0.140	0.012	
 T3	40.96 ± 4.64	40.93 ± 4.41	39.87 ± 4.39	38.08 ± 4.26	P = 0.008, Pa = 0.015, Pb = 0.019	0.225	<0.001	
 I3	45.32 ± 4.41	45.31 ± 4.50	43.93 ± 5.55	42.62 ± 5.39	P = 0.030, Pb = 0.017	0.188	0.001	
 N3	44.52 ± 4.87	44.87 ± 4.47	44.35 ± 4.55	42.42 ± 4.07	P = 0.030	0.199	<0.001	
 S6	34.96 ± 3.00	35.15 ± 2.97	34.86 ± 3.26	33.69 ± 3.82	P = 0.154	0.165	0.003	
 T6	34.95 ± 3.56	35.42 ± 3.15	34.58 ± 3.34	33.31 ± 3.64	P = 0.058	0.159	0.004	
 I6	36.69 ± 4.10	36.81 ± 4.01	36.25 ± 3.84	35.85 ± 3.25	P = 0.429	0.086	0.123	
 N6	41.19 ± 3.30	41.60 ± 2.93	40.63 ± 3.34	39.27 ± 3.09	P = 0.003, Pa = 0.005, Pb = 0.006	0.213	<0.001	
mIPL, µm								
 Whole	34.55 ± 2.54	34.69 ± 2.21	34.01 ± 2.31	33.19 ± 1.77	P = 0.003, Pa = 0.008, Pb = 0.010	0.213	<0.001	
 Inner ring average	39.59 ± 3.32	39.64 ± 3.00	38.77 ± 2.82	37.63 ± 2.18	P = 0.002, Pa = 0.004, Pb = 0.005	0.232	<0.001	
 Outer ring average	29.51 ± 2.38	29.74 ± 2.06	29.26 ± 2.39	28.76 ± 2.10	P = 0.140	0.119	0.036	
 S3	40.16 ± 3.80	39.99 ± 3.51	39.50 ± 3.01	37.54 ± 2.87	P = 0.005, Pa = 0.002, Pb = 0.016, Pc = 0.021	0.215	<0.001	
 T3	38.91 ± 3.43	38.99 ± 3.06	38.10 ± 2.93	37.15 ± 2.85	P = 0.012, Pa = 0.035	0.189	0.001	
 I3	38.65 ± 3.54	38.48 ± 3.42	37.44 ± 3.81	36.81 ± 3.45	P = 0.046	0.174	0.002	
 N3	40.65 ± 3.89	40.81 ± 3.27	40.04 ± 3.36	39.00 ± 2.94	P = 0.031, Pb = 0.039	0.186	0.001	
 S6	27.68 ± 2.51	27.79 ± 2.31	27.63 ± 2.73	26.81 ± 2.40	P = 0.270	0.138	0.014	
 T6	29.89 ± 2.62	30.22 ± 2.37	29.76 ± 2.47	29.77 ± 2.30	P = 0.696	0.029	0.606	
 I6	28.90 ± 2.98	29.19 ± 2.78	28.58 ± 2.93	28.27 ± 2.22	P = 0.463	0.066	0.242	
 N6	31.56 ± 2.91	31.75 ± 2.57	31.07 ± 2.93	30.19 ± 2.79	P = 0.017, Pa = 0.034	0.157	0.005	
 Age	14.59 ± 6.86	13.99 ± 6.70	14.91 ± 6.50	16.74 ± 5.12	P = 0.251	−0.131	0.014	
 AL	18.10 ± 0.81	18.07 ± 0.68	18.48 ± 0.91	19.98 ± 1.01	P < 0.001, Pd = 0.004, Pa < 0.001, Pe = 0.004, Pb < 0.001, Pc < 0.001	−0.556	<0.001	
AL, axial length; G, global; I, inferior; I3, inferior inner ring; I6, inferior outer ring; N, nasal; N3, nasal inner ring; N6, nasal outer ring; NS, nasal superior; NI, nasal inferior; r, two-tailed Pearson's bivariate correlation coefficient; S, superior; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; SE, spherical equivalent; T, temporal; T3, temporal inner ring; T6, temporal outer ring; TI, temporal inferior; TS, temporal superior.

P values without superscripts are multigroup ANOVA result.

a = P value between hyperopia and high myopia.

b = P value between emmetropia and high myopia.

c = P value between mild/moderate myopia and high myopia.

d = P value between hyperopia and mild/moderate myopia.

e = P value between emmetropia and mild/moderate myopia.

P < 0.05 was considered statistically significant (bold font).

Factors Influencing pRNFL/mGCL/mIPL Thickness

The pRNFL thickness was significantly negatively correlated with age and AL in the superior (r = −0.300, P < 0.001 and r = −0.361, P < 0.001), inferior (r = −0.343, P < 0.001 and r = −0.394, P < 0.001), and nasal (r = −0.545, P < 0.001 and r = −0.496, P < 0.001, respectively) quadrants, whereas in the temporal quadrant, the correlation was positive both with age (r = 0.218, P < 0.001) and AL (r = 0.364, P < 0.001). A significant correlation was also observed between pRNFL thickness and SE, which was positive in the superior (r = 0.217, P < 0.001), inferior (r = 0.291, P < 0.001), and nasal (r = 0.286, P < 0.001) quadrants, and negative in the temporal quadrant (r = −0.270, P < 0.001; see Tables 3, 4, Supplementary Table S1).

In the multiple linear regression analysis, with pRNFL parameters as the dependent variable and age, sex, SE, and AL as independent variables, age, AL, or SE were independent relative factors for almost all pRNFL thickness parameters. In the age subgroup multiple regression analysis, SE or AL was significantly associated with pRNFL thickness in almost all sectors in the adult and geriatric groups (Supplementary Table S2).

In Pearson's correlation analysis, mGCL/mIPL thickness was negatively correlated with age (P < 0.05) in all areas of the outer ring; negatively correlated with AL in all regions except S3 and T3, with the strongest correlation in the outer ring (mGCL and outer ring A: r = −0.393, P < 0.001; and mIPL and outer ring A: r = −0.324, P < 0.001); and positively correlated with SE in all areas except I6/T6 (P < 0.05; see Tables 3, 4, Supplementary Table S1). With mGCL/mIPL parameters as the dependent variable and age, sex, SE, and AL as independent variables, the mGCL/mIPL thickness was significantly associated with SE (positively) and sex in the inner ring regions, and significantly negatively associated with AL in the outer ring regions (Table 5).

Table 5. Multiple Linear Regression Analyses Among the Thickness of Peripapillary Retinal Nerve Fiber Layer (pRNFL), Macular Ganglion Cell Layer (mGCL), Macular Inner Plexiform Layer (mIPL), and Ocular and Systemic Parameters

Dependent Variable	Independent Variables Included in Equation*	R 2	β	P Value	
pRNFL					
 G	Age	0.176	−0.350	<0.001	
	SE		0.206	<0.001	
 S	Age	0.115	−0.125	0.044	
	AL		−0.260	<0.001	
 N	Age	0.345	−0.418	<0.001	
	AL		−0.176	0.008	
	SE		0.143	0.009	
 I	AL	0.159	−0.174	0.022	
	Age		−0.193	0.002	
	SE		0.156	0.013	
 T	AL	0.134	0.370	<0.001	
 TS	Age	0.066	−0.263	<0.001	
 NS	AL	0.132	−0.367	<0.001	
 TI	Age	0.088	−0.301	<0.001	
 NI	SE	0.117	0.231	<0.001	
	AL		−0.162	0.009	
mGCL					
 Whole	AL	0.029	−0.179	0.002	
 Inner ring average	SE	0.051	0.186	0.001	
	Sex		−0.150	0.007	
 Outer ring average	AL	0.153	−0.395	<0.001	
 S3	SE	0.017	0.129	0.023	
 T3	SE	0.077	0.212	<0.001	
	Sex		−0.193	<0.001	
 I3	SE	0.041	0.175	0.002	
	Sex		−0.128	0.021	
 N3	SE	0.051	0.188	0.001	
	Sex		−0.147	0.008	
 S6	AL	0.101	−0.322	<0.001	
 T6	AL	0.148	−0.388	<0.001	
 I6	AL	0.051	−0.233	<0.001	
 N6	AL	0.129	−0.363	<0.001	
mIPL					
 Whole	AL	0.061	−0.254	<0.001	
 Inner ring average	SE	0.055	0.219	<0.001	
	Sex		−0.115	0.037	
 Outer ring average	AL	0.096	−0.314	<0.001	
 S3	SE	0.046	0.199	<0.001	
	Sex		−0.110	0.046	
 T3	SE	0.043	0.182	0.001	
	Sex		−0.127	0.022	
 I3	SE	0.022	0.160	0.004	
 N3	SE	0.038	0.176	0.002	
	Sex		−0.115	0.039	
 S6	AL	0.086	−0.298	<0.001	
 T6	AL	0.037	−0.200	<0.001	
 I6	AL	0.043	−0.214	<0.001	
AL, axial length; G, global; I, inferior; I3, inferior inner ring; I6, inferior outer ring; N, nasal; N3, nasal inner ring; N6, nasal outer ring; NI, nasal inferior; T, temporal; NS, nasal superior; S, superior; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; SE, spherical equivalent; T3, temporal inner ring; T6, temporal outer ring; TI, temporal inferior; TS, temporal superior.

* Adjusted for age, sex (male monkeys as reference), SE, and AL.

In the multiple linear regression analysis between mGCL/mIPL and pRNFL parameters, after adjusting for age, sex, SE, and AL, all pRNFL parameters except those for the temporal quadrant, were independently associated with the inner and outer ring mGCL/mIPL parameters (P < 0.05). Temporal pRNFL thickness was mainly associated with the inner ring mGCL/mIPL thickness (Table 6, Fig. 4). To facilitate comparison for the readers, we have provide a comprehensive analysis of ‘mGCL+mIPL’ data which is detailed in Supplementary Tables S4–S10.

Table 6. Multiple Linear Regression Analyses Among Macular Ganglion Cell Layer (mGCL), Macular Inner Plexiform Layer (mIPL) Thickness, and Peripapillary Retinal Nerve Fiber Layer (pRNFL) Parameters

	pRNFL	
	G*	N*	I*	T*	S*	TS*	NS*	TI*	NI*	
	β	P Value	β	P Value	β	P Value	β	P Value	β	P Value	β	P Value	β	P Value	β	P Value	β	P Value	
mGCL																			
 Whole	0.433	<0.001	0.259	<0.001	0.360	<0.001	0.185	0.003	0.269	<0.001	0.273	<0.001	0.206	0.001	0.228	<0.001	0.255	<0.001	
 Inner ring A	0.392	<0.001	0.227	0.001	0.341	<0.001	0.215	<0.001	0.193	0.001	0.211	<0.001	0.153	0.011	0.258	<0.001	0.203	0.001	
 Outer ring A	0.397	<0.001	0.237	<0.001	0.323	<0.001	0.128	0.028	0.310	<0.001	0.287	<0.001	0.226	<0.001	0.227	<0.001	0.214	<0.001	
 S3	0.381	<0.001	0.183	0.009	0.321	<0.001	0.192	0.002	0.231	<0.001	0.258	<0.001	0.175	0.004	0.227	<0.001	0.208	<0.001	
 T3	0.405	<0.001	0.206	0.002	0.362	<0.001	0.227	<0.001	0.209	<0.001	0.233	<0.001	0.139	0.018	0.273	<0.001	0.220	<0.001	
 I3	0.293	<0.001	0.137	0.046	0.270	<0.001	0.135	0.027	0.196	0.001	0.188	0.001	0.174	0.004	0.261	<0.001	0.118	0.048	
 N3	0.386	<0.001	0.221	0.001	0.345	<0.001	0.201	0.001	0.174	0.003	0.186	0.001	0.146	0.015	0.226	<0.001	0.239	<0.001	
 S6	0.370	<0.001	0.236	<0.001	0.306	<0.001	0.073	0.219	0.307	<0.001	0.291	<0.001	0.245	<0.001	0.128	0.022	0.275	<0.001	
 T6	0.446	<0.001	0.241	<0.001	0.376	<0.001	0.200	0.001	0.315	<0.001	0.309	<0.001	0.215	<0.001	0.276	<0.001	0.242	<0.001	
 I6	0.204	0.001	0.144	0.037	0.149	0.013	0.052	0.394	0.214	<0.001	0.177	0.002	0.167	0.005	0.149	0.009	0.059	0.320	
 N6	0.376	<0.001	0.220	0.001	0.285	<0.001	0.114	0.052	0.298	<0.001	0.284	<0.001	0.231	<0.001	0.212	<0.001	0.179	0.002	
mIPL																			
 Whole	0.465	<0.001	0.284	<0.001	0.361	<0.001	0.176	0.004	0.304	<0.001	0.314	<0.001	0.229	<0.001	0.253	<0.001	0.239	<0.001	
 Inner ring A	0.457	<0.001	0.268	<0.001	0.363	<0.001	0.225	<0.001	0.253	<0.001	0.269	<0.001	0.199	0.001	0.284	<0.001	0.208	<0.001	
 Outer ring A	0.367	<0.001	0.241	<0.001	0.273	<0.001	0.105	0.083	0.288	<0.001	0.275	<0.001	0.213	<0.001	0.169	0.003	0.200	0.001	
 S3	0.465	<0.001	0.235	0.001	0.383	<0.001	0.201	0.001	0.287	<0.001	0.319	<0.001	0.220	<0.001	0.258	<0.001	0.260	<0.001	
 T3	0.448	<0.001	0.262	<0.001	0.352	<0.001	0.285	<0.001	0.260	<0.001	0.259	<0.001	0.194	0.001	0.305	<0.001	0.179	0.002	
 I3	0.387	<0.001	0.244	<0.001	0.304	<0.001	0.188	0.002	0.266	<0.001	0.253	<0.001	0.221	<0.001	0.304	<0.001	0.120	0.043	
 N3	0.403	<0.001	0.198	0.004	0.341	<0.001	0.217	<0.001	0.201	0.001	0.230	<0.001	0.161	0.008	0.253	<0.001	0.210	<0.001	
 S6	0.365	<0.001	0.269	<0.001	0.280	<0.001	0.086	0.151	0.281	<0.001	0.277	<0.001	0.215	<0.001	0.110	0.053	0.261	<0.001	
 T6	0.427	<0.001	0.248	<0.001	0.311	<0.001	0.227	<0.001	0.319	<0.001	0.298	<0.001	0.249	<0.001	0.247	<0.001	0.183	0.002	
 I6	0.244	<0.001	0.151	0.029	0.207	0.001	0.097	0.113	0.215	<0.001	0.190	0.001	0.154	0.010	0.167	0.004	0.117	<0.001	
 N6	0.328	<0.001	0.183	0.006	0.222	<0.001	0.111	0.061	0.254	<0.001	0.260	<0.001	0.188	0.001	0.183	0.001	0.125	0.030	
G, global; I, inferior; I3, inferior inner ring; I6, inferior outer ring; N, nasal; N3, nasal inner ring; N6, nasal outer ring; NI, nasal inferior; NS, nasal superior; S, superior; S3, superior inner ring, 1–3 mm from the central fovea; S6, superior outer ring, 3–6 mm from the central fovea; T, temporal; T3, temporal inner ring; T6, temporal outer ring; TI, temporal inferior; TS, temporal superior.

* Adjusted for age, sex (male monkeys as reference), SE, AL, and pRNFL thickness parameters as independent variables, and mGCL/mIPL parameters as the dependent variable.

Discussion

This cross-sectional study provided normative profiles for pRNFL/mGCL/mIPL thickness in healthy cynomolgus macaques aged 3 to 26 years. The distribution and variance tendency of pRNFL/mGCL/mIPL thickness in this study were in accordance with previous findings in humans.21–25

pRNFL/mGCL/mIPL Thickness Profiles

The mean global pRNFL thickness in our study was 95.06 ± 9.42 µm, which is consistent with previously reported findings in cynomolgus macaques, but smaller than that in rhesus monkeys (Supplementary Table S3). In a study26 including 722 rhesus monkeys (age = 10–25 years; 338 male monkeys and 384 female monkeys), the global pRNFL thickness was 102.7 ± 8.5 µm, whereas in another study27 including 44 healthy rhesus monkeys aged 1 month to 23 years, the mean pRNFL thickness was 124.1 ± 8.89 µm. All these measurements were obtained using spectral domain OCT (SD-OCT). These discrepancies in pRNFL thickness between cynomolgus and rhesus macaques may be due to the larger body size and longer AL of rhesus monkeys.28,29 In a study by Kiely et al.,30 they indicated that the AL of cynomolgus macaques is slightly shorter than that of rhesus monkeys in all age groups. However, the mean global pRNFL thickness observed in our study closely aligns with findings from human studies. For example, Alasil et al.21 reported a mean global pRNFL thickness of 97.3 ± 9.6 µm among 190 healthy participants aged 9 to 86 years.

The pRNFL thickness distribution in our study (greatest inferiorly and superiorly, smaller temporally, and smallest nasally; the ISTN rule) is in agreement with the pRNFL thickness distribution around the optic nerve previously reported in cynomolgus macaques by Wu et al.31 Similar pRNFL thickness distribution was observed in human studies.21,32,33 However, other human34 and monkey35 studies reported that normal pRNFL thickness values follow the ISTN rule, with decreasing pRNFL thickness values from the inferior to the temporal quadrant.35 The study by Chen et al.36 found that the ISTN rule was valid for only 43.8% of pRNFL measurements due to the variance of the nasal sector from the expected ISTN pattern.

The distribution of mGCL/mIPL thickness in this study was similar to that in previous human studies,22–25 with greater thickness in the nasal than in the temporal area, and in the inner than in the outer ring (ETDRS).

Compared with the findings of a study22 involving 384 human subjects with an average age of 64.6 ± 9.8 years, the mGCL thickness in the inner ring was slightly smaller in macaques, whereas the mGCL/mIPL thickness in other regions was similar to that in humans. Relative to the findings of another study23 involving 256 self-reported ophthalmologically healthy human subjects aged 20 years and older, the inner ring mGCL thickness was slightly smaller, and the mIPL thickness was slightly greater in macaques than in humans, whereas the mGCL/mIPL thickness in the outer ring was similar to that in humans. Both these studies used SD-OCT (Heidelberg, Germany). Similar results were found in other studies.25

Correlation Among Age, Sex, SE, and AL, and pRNFL/mGCL/mIPL Thickness

In our study, sex-based differences in pRNFL thickness were observed only in the NS sector, in which pRNFL thickness was greater in male monkeys than in female monkeys. Wu et al.31 reported a significantly greater pRNFL thickness in female monkeys than in male monkeys in the inferior quadrant (P = 0.001), and in male monkeys than in female monkeys in the nasal quadrant (P = 0.035). Pasquale et al. did not find any sex-based differences in pRNFL thickness in rhesus monkeys. Similarly, previous human studies have reported no significant sex-based differences in pRNFL measurements.37–40

We observed a positive correlation of temporal pRNFL thickness with age and AL, and a negative correlation with SE. In other quadrants, pRNFL thickness was negatively correlated with age and AL, but positively correlated with SE. This trend of opposing changes in temporal thickness compared with other quadrants has been reported in the study of Pasquale et al.26 and numerous human studies,39,41–44 supporting the conclusion that the average thickness of the pRNFL in the peripapillary region and non-temporal quadrants negatively correlates with AL. Other studies have also reported pRNFL thinning with increasing AL.41,45,46 Lingham et al.33 reported that longer AL was associated with thinner pRNFL in non-temporal areas and thicker pRNFL temporally. These findings are consistent with an optic disc tilt occurring with increasing AL in myopia. Kim et al.47 speculated that as the AL becomes longer, the retina could be dragged toward the temporal horizon. Young et al.48,49 further revealed that smaller non-temporal pRNFL thickness was associated with a higher degree of myopia. As myopia increases, the optic disc becomes temporally tilted, possibly leading to retinal dragging toward the temporal horizon. This would result in pRNFL thickening in the temporal quadrant and temporal dragging of the superior/inferior peak locations.

In our multivariate analysis, age, AL, and SE were significantly associated with pRNFL thickness, suggesting thinning of the non-temporal pRNFL with increasing age, axial elongation, and higher minus SE, which is consistent with the findings of numerous human studies.24,40,41 This may be partly due to structural changes resulting from the loss of ganglion cells caused by aging.50 It remains unknown, however, at what age this age-related axonal attenuation begins. We found no significant differences between the juvenile and subadult groups. Furthermore, as age increases, the AL progressively increases, causing mechanical stretching and thinning of the retina and refractive status shift from hyperopia to myopia. In our study, 86% of monkeys with high myopia were adult or geriatric, whereas the remaining had refractive errors primarily composed of significant astigmatism. Prior studies9 have shown that an increase in the disc diameter associated with high myopia may lead to a sparser distribution of axons per unit area. Additionally, a temporally converging pRNFL bundle caused by increasing myopia may lead to an increased area of abnormal pRNFL measurements.51 An increasing number of studies48,52,53 assessing pRNFL thickness are focusing on the relationship between the degree of myopia and the neuroretinal rim (NR) to avoid misdiagnosis of glaucoma. In a study evaluating the NR in healthy children with myopia, compared with standard RNFL measurements, Bruch's membrane opening-minimum rim width (BMO-MRW) and RNFL measurements acquired using the anatomic positioning system (APS-RNFL) were less affected by the refractive error degree, suggesting that using BMO-MRW and APS-RNFL assessments could more effectively enable avoiding misdiagnoses of glaucoma.

The negative correlation of mGCL/mIPL thickness with age and AL, and the positive correlation with SE in cynomolgus macaques found in this study aligns with the findings of previous human studies.46,54,55 However, previous human studies have reported age-related mGCL/mIPL thinning throughout the entire ETDRS ring, likely due to age-related loss of retinal ganglion cells and their axons,56 as well as the increase in AL with age leading to eye wall expansion. In this study, age-related mGCL/mIPL thinning in macaques was found only in the outer ring. This may be due to the smaller size of macaque eyes compared with that of human eyes, resulting in differences in the arrangement and density of ganglion cells. A biological explanation is still lacking. An alternative reason may be optical effects during OCT imaging caused by the shorter AL of macaque eyes. The reported mean AL of 18.39 ± 1.02 mm in this study is similar to that in previous studies on macaques. For example, Xue et al.57 reported a mean AL of 18.40 ± 1.03 mm in 341 macaques aged 0.5 to 23 years measured using the IOLMaster 700.

Correlation Between pRNFL and mGCL/mIPL Thickness

Previous human studies58 have reported correlations between mGCL/mIPL and pRNFL parameters. Our study provides the first data on the quantitative relationship between SD-OCT-measured mGCL/mIPL and pRNFL parameters in a large sample of healthy cynomolgus macaques. We observed a correlation between mGCL/mIPL and pRNFL parameters except for the temporal region, in which pRNFL thickness was mainly correlated with the inner ring mGCL/mIPL thickness. After adjusting for relevant confounding factors, we further found an association between non-temporal pRNFL thickness and mGCL/mIPL thickness in various regions, with temporal pRNFL thickness being mainly moderately associated with the inner ring mGCL/mIPL thickness. Human studies58 have also reported a weaker correlation between temporal pRNFL and mGCL/mIPL thickness compared with that for other quadrants.

Limitations

First, due to the cross-sectional design, we could not study changes over time. Meanwhile, due to the objective facts of the monkey factory, there are still some deficiencies in sex distribution. However, a further follow-up study plan is being refined and is scheduled to be conducted in 1 year.

Conclusions

The present study is the first to present normative SD-OCT data for pRNFL/mGCL/mIPL thickness in healthy cynomolgus macaques. Moreover, we analyzed the relationship between pRNFL and mGCL/mIPL thickness, as well as their associations with age, sex, SE, and AL. Our findings are similar to those in humans, highlighting the anatomic similarities and the value of the nonhuman primate research model for studying optic nerve diseases.

Supplementary Material

Supplement 1

Acknowledgments

Supported by the National Key R&D Project of China (2020YFA0112701), the National Natural Science Foundation of China (82171057), the Science and Technology Program of Guangzhou, China (202206080005), and the Major Science and Technology Project of Zhongshan City (2022A1007).

Disclosure: Y. Zhang, None; G. Ye, None; K. Chen, None; S. Huang, None; R. Xie, None; J. Chen, None; W. Liu, None; Z. Wang, None; R. Luo, None; J. Zhan, None; Y. Zhuo, None; Y. Li, None; Y. Zhu, None
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