Macular Thickness and Its Associated Factors in a Healthy Cynomolgus Colony: The Non-Human Primates Eye Study

Abstract

Purpose

The research aims to investigate the normal macular thickness range in a healthy cynomolgus colony and examine its association with relevant parameters.

Design

Cross-sectional observational study.

Subjects

A total of 418 healthy cynomolgus monkeys (mean age, 13.22 ± 7.86 years; range, 1–29 years) from the Non-Human Primates Eye Study conducted between 2021 and 2022.

Methods

All included monkeys underwent comprehensive ocular measurements, including macular thickness measurement using spectral-domain OCT. To account for the correlation between eyes, generalized linear mixed models were applied. To assess the gender differences in macular thickness and volume, independent T-tests were employed. Linear regression, including quadratic terms (Age²), was used to examine the relationships between macular subregions and age. Additionally, univariate analyses and multivariate linear regression were conducted to evaluate the associations between the central, average inner ring, and average outer macular thickness and various systemic and ophthalmic parameters.

Main Outcome Measures

Macular thickness in ETDRS subfields and its associations with age and ocular parameters.

Results

Sex statistically significant differences were observed only in the superior sector of the inner ring and in the temporal sector of the outer ring after adjustment. In inner rings, all 4 subfields exhibited a significant age-related decline; meanwhile, temporal and superior subfields decreased with increasing age in the outer ring. After adjustment, macular thickness was independently associated with age and end-of-anesthesia intraocular pressure (IOP) in the central region, axial length (AXL) in the average inner ring, AXL and mid-anesthesia IOP in the average outer ring, and AXL and end-of-anesthesia IOP for the overall average thickness.

Conclusions

This study provided valuable reference data on macular thickness in different ages and sexes of cynomolgus which could be utilized in preclinical nonhuman primate experiments.

Financial Disclosure(s)

Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

As of 2010, the number of individuals with age-related macular degeneration (AMD) had reached 23 million.¹ Meanwhile, AMD is the leading cause of blindness among people >50 years of age in the United States.² It is therefore important to focus on the prevention and treatment of age-related macular diseases in an aging society. The advent of anti-VEGF agents has advanced the treatment of neovascular AMD, resulting in a reduction in blindness caused by neovascular AMD.3, 4, 5 The treatment development work currently focuses on non-neovascular AMD.⁶ Therapeutic options are becoming increasingly diverse with advances in research technologies, such as nanotherapeutics, port delivery systems, radiation therapy, gene therapy, and cell therapy.4, 5, 6 The safety and efficacy of these new treatments are evaluated in preclinical animal studies before proceeding to phase I and II trials. As the most common research animal, mice cannot be employed in macular preclinical studies due to the absence of a macula in mice eyes. In contrast, the ocular anatomy of nonhuman primates (NHPs) is extremely similar to that of humans.^7,8 Meanwhile, NHPs share 90% of their DNA sequences with humans, reflecting their close evolutionary relationship.^9,10 Consequently, NHPs have emerged as the most valuable experimental model for preclinical ocular research, providing valuable insights into both anatomical and genetic aspects of eye-related studies.

Evidence has demonstrated that the alterations of macular thickness are closely correlated with various eye disorders, optic neuropathy, diabetic retinopathy, and retinal vein occlusion, besides AMD.^11,12 Exploring the normal range of macular thickness plays an important role in monitoring and diagnosing a wide range of retinal diseases. Recognized as one of the parameters in the evaluation of many retinal disorders, the normal range of macular thickness plays a significant role in clinical assessment. Meanwhile, relevant human macular parameters do not provide an equivalent reference for preclinical investigations in NHPs because of species differences.

Current studies involving NHPs have generally relatively small sample sizes and limited age spans, which may not provide sufficiently robust evidence to accurately reflect actual conditions. Therefore, establishing the reference value of NHP macular thickness is essential to serve as a standard.

Spectral or Fourier domain OCT, known for its quantitative capability and higher sensitivity,¹³ can measure subtle changes in macular thickness compared with traditional methods such as fluorescein angiography and time-domain OCT.14, 15, 16 Thus, spectral-domain OCT has been widely applied in clinical trials and scientific research,17, 18, 19, 20 and it is the most commonly used equipment in scientific studies of NHPs.^21,22

This study involved 418 healthy monkeys, ranging in age from 1 to 29 years. The purpose was to analyze the normal range and associated parameters of normal macular thickness in cynomolgus monkeys, aiming to provide a reference for preclinical macular research of NHPs.

Methods

Animals and Ethnic Statement

Nonhuman primates (Macaca fascicularis, cynomolgus) involved in this study were from Huazhen Bioscience, located in Guangzhou, which specializes in breeding monkeys for use as animal models in preclinical studies of biopharmaceuticals. All NHPs in this study grew naturally without any invasive or biomedical interventions, including drug treatment, adeno-associated virus intervention, or surgical intervention related to preclinical experiments. Receiving regular 12-h sunlight to maintain a normal circadian rhythm, macacas were housed in a dedicated feeding facility maintained at approximately 16 °C to 26 °C temperature and approximately 40% to 70% room humidity. Fresh water, fruits, and primate chow (12% of calories from fat, 18% from protein, and 70% from carbohydrates, 200–300 g/d) were provided ad libitum to cynomolgus monkeys to meet their nutritional needs. Music and toys were supplied for entertainment. Furthermore, their physical condition was continuously monitored by skilled veterinarians.

The Huazhen Bioscience has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care to perform animal scientific research in China. All procedures in this study complied with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research and adhered to the Declaration of Helsinki. The study was approved by the Ethical Committee of the Guangzhou Huazhen Biosciences Company (Ethics Number: 2020-168).

Standardized Examination Process

Detailed examination procedures have been described in our published paper.¹⁰ All included monkeys underwent the standardized systemic and comprehensive ocular examination. After administration of an appropriate dose of anesthetic, the animals were fixed in the proper position. Body weight and intraocular pressure (IOP) were recorded; meanwhile, slit lamp examination, fundus photography, A-scan ultrasonography examination, anterior segment OCT, and spectral-domain OCT images were performed. Throughout the entire examination, respiration and heart rate were monitored at all times.

Ocular Examination

After being intramuscularly (IM) anesthetized with compound mixed tiletamine (4 mg/kg body weight, Virbac) and ketamine (0.2 mg/kg body weight, Sumianxin, Shengda Animal Medicine), the monkeys were stabilized in a supine position on a soft flat work desk by a skilled technician. The Icare tonometer (TA01i, Icare) was used to measure IOP. By holding the Icare tonometer to the monkey's pupil and keeping the monkey's head in gaze, 6 measurements were automatically obtained. To ensure data availability, this procedure was repeated 3 times, and the mean IOP was calculated. The IOP of all subjects was measured at the beginning, middle, and end of anesthesia. The anterior segment structure of cynomolgus monkeys was carefully examined by an experienced ophthalmologist using silt lamp biomicroscopy (TOPCON Slit-lamp SL-D701), such as the eyelids, anterior chamber, and cornea. A-scan ultrasonography (TOMEY AL–4000) was used to measure lens thickness, AXL, and anterior chamber depth. Autorefraction and corneal radius of curvature were tested by the Fario FXR–710 (Fario), and spherical equivalent was calculated automatically by this autorefractor. The anterior-segment OCT (Heidelberg Engineering GmbH) was used to measure the anterior chamber and its angle.

Macular Thickness Measurement by Spectral-Domain OCT

The monkeys were anesthetized, and the 0.5% tropicamide phenylephrine hydrochloride eye drop was utilized for pupil dilation. An experienced operator used the machine (SPECTRALIS HRA + OCT, Heidelberg Engineering GmbH) to focus on the pupil first and readjust the camera to focus on the macular fovea, then chose the “volume scan” and acquired images with 49 line B-scan and 20 automated real-time tracking frames. The built-in software automatically generated the macular thickness maps. After completing the OCT measurement, the relevant machine parameters have been set. After finding the thickness map tab in the menu bar, the 1-, 3-, and 6-mm ETDRS has been chosen to get the data. The map consisted of 3 concentric circles and was divided into 9 sectors (the central macular areas, the inner ring, and the outer ring) following the definition of the ETDRS. Both the inner and outer rings were further subdivided into the nasal, temporal, superior, and inferior quadrants. Furthermore, we used the software's divider for automatic image segmentation, and manually corrected the automatic segmentation when a problem occurred. The “Thickness Map Single Exam Report” provided thickness information. Images with inadequate quality or with a signal strength index of <15 decibels (as recommended by the manufacturer) were excluded from analysis.

Inclusion and Exclusion

In this study, none of the cynomolgus monkeys received any biomedical interventions, and all included female monkeys were not pregnant, avoiding the drug effects and the effects of anesthetics on the fetus. The OCT images were carefully reviewed to exclude images with poor quality, such as images with indistinct features or artifacts. After data and OCT image quality control, as well as review of the fundus photographs, 34 eyes were excluded due to the ocular diseases affecting macular thickness, including retinal vein occlusion and hypertensive retinopathy. Additionally, 3 individuals with suspected spontaneous glaucoma and 50 individuals with high myopia (spherical equivalent < –5.00 diopters) were excluded. The definition of glaucoma was according to the recommendations of the Association of International Glaucoma Societies.²³ Ultimately, 836 eyes were included for data analysis (Fig 1).

Figure 1.

Flow diagram showing the inclusion criteria for cynomolgus monkeys and the reasons for exclusion.

Statistical Analysis

Data from each normal eye were included in the analysis. All statistical analyses were performed using the Statistical Package for the Social Sciences version 25.0 (SPSS Inc). Normally distributed data were shown by mean and standard deviation. Average macular thickness across regions and quadrants was compared between females and males using T-tests. To account for correlation between both eyes, potential confounding factors, including age, body weight, and AXL, generalized linear mixed models (GLMMs) were subsequently performed following the t-tests. Quadratic terms of age were included in linear regression analyses to explore the relationship between age and thickness in each macular subregion. Univariate analysis and multiple linear regression analyses, as well as GLMM, were utilized to assess the correlation between the macular thickness and relevant factors.

Results

A total of 418 monkeys were included in this analysis (253 female and 165 male). Table 1 provides a detailed comparison of basic demographic data and ocular parameters of the 2 groups in the included and excluded subjects. The mean age and weight of included monkeys were 13.22 ± 7.86 years old and 4.20 ± 1.33 kg, respectively. No significant statistical differences were observed between the 2 groups in terms of body weight or sex distribution. Compared with the excluded group, the included group was younger and had lower IOP, shorter AXL (P < 0.001), spherical equivalent (P < 0.001), anterior chamber depth (P < 0.001), and thinner central corneal thickness (P = 0.011).

Table 1.

Comparisons of Baseline Characteristics between the Included and Excluded Groups

Parameters	Included Group	Excluded Group	$\mathit{t}/{\mathit{x}}^{\mathbf{2}}$	P Value
Total, N	418	70
Age (yrs)	13.22 ± 7.86	17.49 ± 5.42	–5.65	<0.001
Weight (kg)	4.20 ± 1.33	4.54 ± 1.64	–1.75	0.081
Gender (female %)	253 (60.52)	165 (39.47)	1.63	0.202
IOP (mmHg)
At the beginning of anesthesia	23.41 ± 3.46	25.08 ± 3.74	–5.10	<0.001
In the middle of anesthesia	20.15 ± 5.02	21.43 ± 4.65	–2.75	0.006
At the end of anesthesia	18.02 ± 3.98	19.45 ± 4.49	–3.46	0.001
CCT	453.69 ± 38.15	465.45 ± 51.38	–2.56	0.011
AXL (mm)	18.11 ± 0.86	19.42 ± 1.14	–12.89	<0.001
SE (D)	–0.45 ± 1.90	–6.21 ± 4.94	13.57	<0.001
ACD (mm)	3.03 ± 0.39	3.22 ± 0.33	–6.78	<0.001

ACD = anterior chamber depth; AXL = axial length; CCT = central corneal thickness; D = diopters; IOP = intraocular pressure; SE = spherical equivalent.

Values for mean ± standard deviation or total number (percentage) are shown. Statistically significant P values (P < 0.05) are in boldface.

Table 2 presents the gender differences in macular thickness and volume in the selected healthy cynomolgus colony. The average macular thickness of the cohort was 333.91 ± 28.01 μm, with the average values of 343.04 ± 25.72 μm for the inner ring and 343.12 ± 27.08 μm for the outer ring. In both males and females, the central macular region was thinnest, measuring 322.24 ± 39.17 μm and 323.91 ± 44.27 μm, respectively. In addition, within both the inner and outer rings, the temporal subfield was the thinnest, and the nasal subfield was the thickest. The distribution of macular thickness by sex is shown in Figure 2. Statistically significant differences were observed only in the superior sector of the inner ring (adjusted P values = 0.006) and in the temporal sector of the outer ring (adjusted P values = 0.012) after using GLMM.

Table 2.

The Difference in Macular Thickness and Volume by Gender

Macular Thickness (μm)	Female	Male	P Value	Adjusted P Value	G
N	253	165
Central macula	323.91 ± 44.27	322.24 ± 39.17	0.588	0.509	323.18 ± 42.30
Inner ring
Temporal	321.38 ± 35.22	335.03 ± 27.47	<0.001	0.145	326.66 ± 33.09
Superior	343.43 ± 31.42	352.54 ± 27.02	<0.001	0.006	346.96 ± 30.10
Nasal	348.40 ± 32.59	357.51 ± 25.75	<0.001	0.078	352.70 ± 27.78
Inferior	335.40 ± 34.84	345.47 ± 31.57	<0.001	0.231	339.29 ± 33.94
Average inner ring	336.98 ± 27.46	347.63 ± 21.28	<0.001	0.912	343.04 ± 25.72
Outer rings
Temporal	327.66 ± 28.16	339.20 ± 23.41	<0.001	0.012	332.13 ± 26.99
Superior	345.84 ± 40.42	349.47 ± 27.71	0.168	0.968	347.24 ± 36.07
Nasal	349.89 ± 29.68	357.12 ± 23.88	<0.001	0.403	353.93 ± 28.98
Inferior	342.96 ± 30.92	348.11 ± 37.32	0.035	0.214	344.95 ± 33.62
Average outer ring	340.34 ± 28.99	347.53 ± 23.09	<0.001	0.899	343.12 ± 27.08
Average macular thickness	321.71 ± 65.44	318.00 ± 80.37	0.466	0.893	333.91 ± 28.01
Macular volume	3.23 ± 0.56	3.25 ± 0.25	0 0.482	0.319	3.23 ± 0. 47

G: the average values for the female and male. Statistically significant P values (P < 0.05) are shown in bold. P value represents the result of the unadjusted t-test. Adjusted P values were derived from generalized linear mixed models after controlling for intereye correlation, age, body weight, and axial length.

Figure 2.

The distribution of macular thickness by gender. RPE = retinal pigment epithelium.

Table 3 presents the distribution of macular thickness (including 9 subfields) and volume measurements by age, ranging from 1 to 29. The subjects were categorized into 4 age groups: juvenile (1–4 years), subadult (4–9 years), adult (9–20 years), and geriatric (20∼29 years).²⁴ Significant age-related variations were observed in most quadrants, showing a trend with increasing age. Central macular thickness increased from the juvenile group (309.75 ± 37.69 μm) and the subadult group (307.83 ± 39.80 μm) to the adult group (336.55 ± 45.28 μm), and then decreased in the geriatric group (326.36 ± 34.83 μm). Quadratic regression confirmed a significant inverted U-shaped relationship (Age² B = –0.18, 95% confidence interval –0.29 to –0.09; P = 0.001). In inner rings, all 4 subfields exhibited a significant age-related decline (P < 0.05); meanwhile, temporal and superior subfields decreased with increasing age in the outer ring (Fig S1, available at www.ophthalmologyscience.org). Furthermore, compared with Table 2, the nasal subfield remained the thickest and the temporal subfield the thinnest quadrant in both inner and outer rings.

Table 3.

Macular Thickness and Volume Measurements by OCT in NHPs by Age Group

Macular Thickness (μm)	Age Group (yr)				Trend for Age
	1∼4 Juvenile	4–9 Subadult	9–20 Adult	20∼29 Geriatric	Age/Age2 B (95% CI)	Age/Age2 P Value
No. of subjects	84	87	160	87
Central macula∗	309.75 ± 37.69	307.83 ± 39.8	336.55 ± 45.28	326.36 ± 34.83	–0.18 (–0.29,–0.09)	0.001
Inner rings
Temporal	342.21 ± 24.63	330.88 ± 31.94	321.53 ± 36.82	316.51 ± 27.83	–1.17 (–1.45,–0.89)	<0.001
Superior	354.69 ± 26.94	349.83 ± 26.44	343.82 ± 33.03	342.22 ± 29.20	–0.63 (–0.89,–0.37)	<0.001
Nasal	361.96 ± 27.15	354.51 ± 24.96	349.01 ± 35.67	344.96 ± 25.33	–0.78 (–1.04,–0.51)	<0.001
Inferior	349.95 ± 28.58	337.80 ± 36.39	338.62 ± 35.29	330.97 ± 32.26	–0.63 (–0.93,–0.34)	<0.001
Outer rings
Temporal	342.11 ± 23.91	340.70 ± 22.59	324.88 ± 29.51	326.48 ± 23.77	–1.04 (–1.27,–0.82)	<0.001
Superior	352.55 ± 31.53	355.30 ± 22.64	345.63 ± 45.74	337.05 ± 27.49	–0.18 (–1.13,–0.51)	<0.001
Nasal∗	357.69 ± 23.06	360.16 ± 23.17	349.96 ± 31.82	345.42 ± 25.76	–0.04 (–0.09,–0.01)	0.152
Inferior∗	340.79 ± 36.03	354.67 ± 40.58	343.69 ± 30.73	342.40 ± 33.06	–0.08 (–0.14,–0.06)	0.160
Volume (mm3)	3.25 ± 0.16	3.23 ± 0.17	3.18 ± 0.32	3.15 ± 0.32	–0.002 (–0.006,0.002)	0.282

CI = confidence interval; NHPs = nonhuman primates.

Parameters marked with ∗indicate that a quadratic term (Age²) was included in the regression model to assess for nonlinear associations. Statistically significant P values (P < 0.05) are in boldface.

Building on the age group analysis, we further explored the associations between demographic and ocular parameters and macular thickness. Univariate analyses were first conducted to preliminarily assess the relationships (Table S1, available at www.ophthalmologyscience.org).

Parameters that showed statistical significance were then included in multiple linear regression and GLMM analyses. The results are shown in Tables 4 and 5. For central macular thickness, age (P < 0.001) and IOP at the end of anesthesia (P = 0.001) remained significant predictors after adjustment. In the inner ring, AXL (P = 0.002) was independently correlated with thickness, while IOP in the middle of anesthesia (P = 0.438) lost statistical significance after adjustment using the GLMM model. For the outer ring, AXL (P < 0.001) and IOP measured in the middle of anesthesia (P = 0.025) were independently associated with average thickness after GLMM adjustment. Overall average macular thickness was independently associated with AXL (P < 0.001) and IOP measured at the end of anesthesia (P = 0.048).

Table 4.

Multiple Linear Regression Analysis of Ocular Factors Associated with Macular Thickness

Macular Regions	Parameters	β (B)	95% CI	P Value
Central macula	Age	1.02	0.63–1.41	<0.001
	IOP at the end of anesthesia	1.37	0.59–2.16	0.001
Average inner ring	AXL	–23.17	–29.50 to 16.84	0.002
	IOP in the middle of anesthesia	1.28	0.34–2.22	0.007
Average outer ring	Age (years)	–0.90	–1.58 to –0.22	0.010
	IOP in the middle of anesthesia	0.87	0.13–1.61	0.020
	AXL	–13.22	–18.40 to –8.05	<0.001
	ACD	13.71	2.23–25.19	0.019
Overall average macula	IOP in the middle of anesthesia	0.85	0.02–1.69	0.045
	IOP in the end of anesthesia	1.11	0.08–2.15	0.034
	AXL	–10.95	–14.91 to –6.98	<0.001

ACD = anterior chamber depth; AXL = axial length; CI = confidence interval; IOP = intraocular pressure.

Statistically significant P values (P < 0.05) are in boldface.

Table 5.

Generalized Linear Mixed Model Analysis of Ocular Factors Associated with Macular Thickness

Macular Regions	Parameters	β (B)	95% CI	P Value
Central macula	Age	1.03	0.57–1.49	<0.001
	IOP at the end of anesthesia	1.26	0.37–2.15	0.005
Average inner ring	AXL	–3.63	–6.24 to –1.02	0.006
	IOP in the middle of anesthesia	–0.17	–0.60 to 0.26	0.438
Average outer ring	Age (years)	–0.15	–1.07 to 0.75	0.734
	IOP in the middle of anesthesia	1.12	0.14–2.10	0.025
	AXL	–16.62	–23.32 to –9.92	<0.001
	ACD	2.78	–12.20 to 17.76	0.715
Overall average macula	IOP in the middle of anesthesia	0.87	–0.02 to 1.78	0.056
	IOP in the end of anesthesia	1.12	0.008–2.24	0.048
	AXL	–11.30	–15.61 to –6.98	<0.001

ACD = anterior chamber depth; AXL = axial length; CI = confidence interval; IOP = intraocular pressure.

Statistically significant P values (P < 0.05) are in boldface.

Discussion

A total of 418 cynomolgus monkeys of different ages were included from a healthy breeding colony at Huazhen, South China. This study revealed the normal range of monkey macular thickness across different sexes, regions, and age groups, and further examined the associations between macular thickness and various ocular and systemic factors.

In our study, the average macular thickness was 333.91 ± 28.01 μm. Compared with the research published in 2019,²⁵ the normal range of macular thickness in the temporal inner (327 ± 17 vs. 326.66 ± 33.09 μm) and inferior inner quadrants (339 ± 17 vs. 339.29 ± 33.94 μm) was similar. But in other quadrants, the differences were notable, the macular thickness comparison in other subfields were 341 ± 14 versus 346.96 ± 30.10 μm (superior inner), 341 ± 18 versus 352.70 ± 27.78 μm (nasal inner), 299 ± 20 versus 332.13 ± 26.99 (temporal outer), 320 ± 16 versus 347.24 ± 36.07 μm (superior outer), 332 ± 23 versus 344.95 ± 33.627 μm (inferior outer), and 337 ± 18 μm versus 353.93 ± 28.98 μm (nasal outer). Summing up the results, we found that the thickness of most macular regions was significantly thicker compared to the study performed in 2019, though all the monkeys were cynomolgus monkeys. There are several reasons that might cause these differences.²⁵ First, in the study by Denk et al,²⁵ the subjects age ranged from 20 months to 50 months, which is nearly equivalent to the minimum age of subjects in our study. Several pediatric epidemiological studies showed that the macular thickness would become thicker with aging, which may also happen to young monkeys.26, 27, 28 Second, as a large sample epidemiological study, our research included a wide age range of subjects. Third, the monkeys in the study by Denk et al²⁵ had a Mauritian genetic background. Previous studies have shown that Mauritian cynomolgus macaques, due to a founder effect and long-term island isolation, exhibit markedly reduced genetic diversity and clear differences from mainland Asian populations.^29,30 Therefore, genetic background may also contribute to differences in macular thickness. Supporting this, human studies have reported that macular thickness can vary across ethnic groups.31, 32, 33 However, whether genetic differences between cynomolgus monkey populations indeed influence macular thickness remains to be further investigated and verified.

Compared with the epidemiological research for human macular thickness (Table 6), we found that the thickness of all 9 regions was greater in NHPs than in humans. Moreover, the study shows that the macular volume was approximately 2 times thinner in monkeys than in humans, which might be attributed to the smaller somatotype of monkeys.³⁴ What’s more, the temporal region was thinnest and the nasal region was greatest in the inner ring, whether in monkeys or humans. But in outer rings, our region trends in compliance with some studies^35,36 and in contrast to some studies,³⁷ which suggests that there is still controversy. For gender difference, males showed significantly greater thickness than females in the superior subfield of the inner ring and in the temporal subfield of the outer ring (P = 0.006 and P = 0.012, respectively). The central subfield thickness was slightly greater in females than in males (P = 0.509), consistent with the findings of Denk et al²⁵ in cynomolgus monkeys. However, the central subfield thickness of males was usually thicker than females in humans.^38,39 Although the gender difference in central subfield thickness was not statistically significant in our study, whether the thickness of the macula center in monkeys is different from that in humans needs to be further explored. Furthermore, in our study, the outer rings appeared thicker than the inner rings, which was the opposite of what was observed in humans.^40,41 Given the large sample size and the wide age span of the monkeys, this may result in the relatively large variation influenced by age and the variation directly reflected in the standard deviation of the value. Finally, similar to humans, macular thickness decreases with age in some quadrants. Overall, there is a high degree of similarity between human and monkey macular thickness, although some differences exist. Importantly, these cross-species comparisons are descriptive and should be interpreted with caution, as there is currently no established age translation between monkeys and humans.

Table 6.

Macular Thickness and Volume in Previous Nonhuman Primate and Human Eye Studies

Study	Year	N	Monkey/Human	Age (Year/Month)	OCT	Macular Volume	Central Macula	Inner				Outer
								Inferior	Nasal	Superior	Temporal	Inferior	Nasal	Superior	Temporal
Current study	2023	418	Cynomolgus monkey	4∼29	Heidelberg Engineering	3.23 ± 0. 47	323.18 ± 42.30	339.29 ± 33.94	352.70 ± 27.78	346.96 ± 30.10	326.66 ± 33.09	344.95 ± 33.62	353.93 ± 28.98	347.24 ± 36.07	332.13 ± 26.99
Denk et al25	2019	160	Cynomolgus monkey	20th∼50th	Heidelberg Engineering		244 ± 21	339 ± 17	341 ± 18	341 ± 14	327 ± 17	332 ± 23	337 ± 18	320 ± 16	299 ± 20
Patel et al35	2015	67 321	Human	40∼69	Topcon 3D OCT1000 Mark II	7.87 ± 0.37	264.5 ± 22.9	311.9 ± 16.2	319.5 ± 16.3	315.3 ± 16.1	304.0 ± 15.5	263.0 ± 15.0	287.2 ± 15.8	269.7 ± 14.3	255.6 ± 14.3
Duan42	2010	2230	Human	30∼70+	Stratus OCT, Model 3000	6.761 ± 0.516		260.0 ± 15.5	252.9 ± 19.1	261.6 ± 16.2	246.7 ± 16.7	225.5 ± 13.3	258.514.5	240.0 ± 13.5	227.0 ± 13.1
Myers et al43	2015	4926	Human	43∼86	Topcon 3D-OCT 1000-Mark II			332.9 ± 17.2	341.3 ± 17.9	334.9 ± 15.9	329.0 ± 17.3	281.3 ± 14.7	306.0 ± 17.0	288.3 ± 13.8	283.2 ± 15.1
Gupta et al36	2013	490	Human	40∼80	Cirrus HD-OCT	10.09 (0.41)	250.38 (20.58)	319.09 (14.55)	325.14 (15.77)	323.11 (15.10)	310.00 (14.45)	266.19 (12.66)	299.38 (14.17)	279.62 (13.06)	261.48 (12.21)
Natung et al44	2016	500	Human	>18	Model 500; Carl Zeiss Meditec			313.47 ± 19.14	316.98 ± 19.16	317.16 ± 17.84	303.38 ± 16.24	266.68 ± 22.15	294.46 ± 17.37	277.59 ± 17.83	260.70 ± 14.63
Al-Zamil et al45	2017	158	Human	18–56	Cirrus HD-OCT	8.48 ± 0.35	240.40 ± 18.26	315.94 ± 21.05	320.23 ± 20.71	318.38 ± 21.69	303.81 ± 20.70	265.75 ± 17.31	293.18 ± 21.21	276.37 ± 16.58	259.56 ± 25.24

This research involved 418 monkeys and 836 eyes, which is the largest study about normal NHP macular thickness to date. Moreover, the age distribution of the participants ranged from 1 to 29 years old, providing a comprehensive view of macular thickness in different age groups. These findings offer valuable guidance for selecting monkeys of the appropriate age for various purposes or preclinical experiments. What’s more, because of the high price of NHPs, macula-related studies often include only 1 or 2 animals in the control group, making it difficult to determine whether the macula thickness of the selected monkey is within the normal range. Providing the normal range of macular thickness, our study may help to partially address this problem. However, there are still limitations in our study. First, female monkeys accounted for 60.5% of all subjects. In a concentrated life environment, the natural territorial awareness will make some male monkeys die from fighting. In addition, many biopharmaceutical companies preferentially select young male monkeys for biological experiments. Second, this study is limited by the lack of test–retest validation for macular thickness measurements. Although all operators were trained according to standardized protocols, the absence of repeated measurements may limit the assessment of measurement variability. Third, all measurements were performed under general anesthesia, which may slightly affect IOP and macular thickness. Additionally, all monkeys were from a single breeding colony, which may limit the generalizability of the findings.

In conclusion, our study measured the distribution of macular thickness in a healthy cynomolgus colony and found that the macular thickness of monkeys was thicker than humans. In addition, our findings provided normative reference values for preclinical NHP studies about macular diseases.

Manuscript no. XOPS-D-25-00469.