Molecular Vision 2025; 31:276-282
<http://www.molvis.org/molvis/v31/276>
Received 17 July 2024 | Accepted 26 September 2025 | Published 28 September 2025
Molecular Vision 2025; 31:276-282
<http://www.molvis.org/molvis/v31/276>
Received 17 July 2024 |
Accepted 26 September 2025 |
Published 28 September 2025
Jinan Zhan,1 Zhidong Li,1 Guitong Ye,1 Yuan Zhang,1 Kezhe Chen,1,2 Rui Xie,1 Wei Liu,3 Jian Wu,1,4 Yingting Zhu,1 Yehong Zhuo1
The first two authors contributed equally to this work
1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangdong Provincial Clinical Research Center for Ocular Diseases, Guangzhou, China; 2Jinan University First Affiliated Hospital, Guangzhou, Guangdong, China; 3Huazhen Biosciences, Zuocun Village, Conghua District, Guangzhou, China; 4Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
Correspondence to: Ying-ting Zhu, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangdong Provincial Clinical Research Center for Ocular Diseases, Guangzhou, China; Phone: 8613580585671; email: zhuyt35@mail.sysu.edu.cn
Purpose: To investigate the intraocular pressure (IOP) distribution of cynomolgus monkeys in different age groups after anesthesia and mydriasis.
Methods: A total of 158 young and 252 old cynomolgus monkeys, aged 3.95 (1.61; range, 1.38 to 5.97) and 18.80 (1.67; range, 15.21 to 24.69) years, respectively, were subjected to general anesthesia (intramuscular injection of Zoletil 4 mg/kg mixed with xylazine/ketamine 0.2 mg/kg). IOP was measured using an Icare tonometer at three time points: immediately following the onset of anesthesia, after stabilization but before mydriasis, and after mydriasis (induced by 1% tropicamide eye drops).
Results: No significant differences were observed between sexes or between eyes within the Macaca fascicularis colony. After anesthesia stabilization but before mydriasis, the IOP of the old M. fascicularis colony was 22.33 mm Hg (4.12; range, 12.33 to 37.33), whereas the IOP of the young M. fascicularis colony was 16.67 mm Hg (4.00; range, 10.67 to 25.67). After mydriasis, the IOP of the old M. fascicularis colony was 19.33 mm Hg (5.00; range 11.33 to 33.00), while the IOP of the young M. fascicularis colony was 15.67 mm Hg (4.50; range, 9.67 to 24.00). All measurements were conducted with the monkeys in a prone position. Notably, both the young and old colonies experienced a significant decrease in IOP after mydriasis (p < 0.001). Moreover, the reduction observed in the young colony was significantly greater than that in the old colony (p < 0.001).
Conclusions: The IOP levels of the old M. fascicularis colony were higher than those of the young one, regardless of whether it was after anesthesia, before mydriasis, or after mydriasis. Furthermore, monkeys of different age groups had different reductions in IOP under different interventions (anesthesia, mydriasis). Additionally, within the M. fascicularis colony, IOP was equivalent between males and females, as well as between the right and left eyes.
Intraocular pressure (IOP), the stress exerted on the intraocular structure by the tunics, is a crucial parameter of the eye, which has a well-defined relationship to the balance between aqueous humor production and outflow. Elevated IOP is well recognized as a major risk factor for glaucoma and is also implicated in the pathogenesis of myopia [1]. Therefore, whether it involves screening for glaucoma, elucidating the pathogenesis of myopia, or identifying high-risk populations, the exploration of factors influencing IOP holds significant potential value.
Nonhuman primates and Homo sapiens possess highly conserved sequences and proteins, underscoring the strong genetic connection between the two species. Due to its anatomic, physiologic, and genetic similarities to humans, the nonhuman primate serves as an excellent zoological model for preclinical studies, basic research, and drug experiments, particularly in the field of ophthalmic diseases. In humans, IOP changes nonlinearly with age [2]. In both healthy and diseased animal models, the IOP is variable and influenced by circadian rhythm [3], corneal parameters [4], pupil dilation [5], myopia, and metabolism [6]. These factors are variable in different age groups. Therefore, it is crucial to directly examine the relationship between age and IOP.
Several limited-scale studies have investigated the IOP after general anesthesia and mydriasis in Macaca fascicularis, while anesthesia and mydriasis play a critical role in animal ophthalmic research, but the sample sizes of these investigations are relatively small. In light of this research gap, the present study aims to explore the distribution of IOP in both old and young cynomolgus monkeys following anesthesia within a standardized cynomolgus colony. This study presents the IOP after general anesthesia and mydriasis, which provides a good estimate of normal IOP of cynomolgus macaques.
All procedures involving animals were conducted under protocols approved by the Ethical Committee of the Guangzhou Huazhen Biosciences Company (Ethics Number: 2020-168) and Zhongshan Ophthalmic Center (Permit Number: SYXK (YUE) 2018-0189). In addition, the Association for Research in Vision and Ophthalmology’s statement on the use of animals in ophthalmic and vision research was followed.
This study is based on information gathered from the Non-Human Primates Eye Disease Study [7]. The M. fascicularis used in this study were acquired from Huazhen Animal Laboratory Center, a reputable organization known for breeding and supplying high-quality animals. The monkeys were bred in South China and provided with a well-regulated living environment. The housing environment was maintained from 16 °C to 26 °C, with the relative humidity keep between 40% and 70%, ensuring optimal conditions for this primate species.
To establish a consistent circadian cycle, the monkeys were exposed to 12 h of daylight, followed by 12 h of darkness. They had continuous access to water and were fed a specially formulated diet, comprising 12% fat, 18% protein, and 70% carbohydrates, with a daily intake of 200 to 300 g. In addition, they were provided with fresh fruits, nutritional supplements, and indoor entertainment such as toys and music.
The study included young monkeys under the age of 6 years and old monkeys aged between 15 and 25 years. Based on the social maturity age of male cynomolgus monkeys (5.5 years old) and female cynomolgus monkeys (3.5-4 years old), the age range of 7 to 10 years in monkeys corresponds to young adult humans under 30 years old, and the range of 20 to 26 years in monkeys is equivalent to elderly humans over 70 years old. Therefore, monkeys younger than 6 years were categorized as the young group, and the old group consisted of monkeys aged 15 to 25 years.
Only monkeys without any preexisting ocular or systemic diseases and deemed suitable for anesthesia were included in the study. Any monkeys that exhibited other ocular or systemic diseases during the examination, pregnant or lactating females, and those with adverse reactions to anesthesia were excluded from the study.
Cynomolgus macaques (M. fascicularis), a species within the family Cercopithecidae and genus Macaca, were used in this study. All subjects received anesthesia through an intramuscular injection of Zoletil (4 mg/kg bodyweight; Virbac, Carros, France), mixed with xylazine/ketamine, at a dosage of 0.2 mg/kg (Sumianxin; Shengda Animal Medicine, Zhejiang, China). They maintained a flat posture and a fixed gaze. An eyelid opener was to keep the eyelids open, and all examinations lasted 60 to 120 min according to standardized protocols.
The anesthesia dosage was carefully administered within the established safe range, and the veterinary team ensured continuous monitoring and care of the examination site throughout the procedure. During anesthesia, the monkey group primarily exhibited increased salivation and decreased body temperature; therefore, atropine was administered to keep the monkeys warm. No other adverse reactions were observed during the course of our measurements. Supplementary medication (0.2-0.3 mg/kg) was administered if awakening occurred during the procedure. After the third IOP collection was completed, the monkeys gradually woke up and were given anesthesia for resuscitation. Temperature influences IOP [8], which was only measured at 25 °C in this study. Figure 1 shows the process of measuring IOP.
After a suitable induction period of anesthesia (lasting between 5 and 15 min), the initial IOP measurement (IOP1) was taken using the Icare (TA01; Icare, Helsinki, Finland) rebound tonometer. A total of six IOP measurements were recorded and subsequently averaged. Any measurements where the difference in IOP exceeded 3 mm Hg among the six readings were disregarded and excluded from the analysis. Additional ocular examinations were subsequently performed. After a duration of 29 ± 17 (median ± interquartile range [IQR]) minutes following anesthesia, the second IOP measurement (IOP2) was obtained, and mydriasis was induced. The macaque monkeys were placed in a comfortable head position, and topical 1% tropicamide eye drops (Mydrin; P, Santen Osaka, Japan) were administered twice, each time for 5 min. Following the final administration, a 30-minute waiting period was observed, during which the light reflex was evaluated. A pupil dilation greater than 6 mm was considered sufficient and indicated the initiation of the subsequent examinations. However, if this criterion was not met, an additional drop of the eye drops was administered. Subsequently, a waiting period was observed for complete dilation. After a period of 92 ± 30 (median ± IQR) min from the onset of anesthesia, the third IOP measurement (IOP3) was performed.
We generated all calculations and graphs using R, version 4.2.2 (R Core Team, Vienna, Austria). For variables that followed a normal distribution, descriptive statistics were reported as mean ± standard deviation, and a t test was used for two-group comparisons. For variables that did not fit a normal distribution, the median (IQR) was used for description. Group comparisons were performed using the Mann-Whitney U test. Given the small sample size, the Shapiro-Wilk test was used for normality test. The p values less than 0.05 (two-tailed) were deemed statistically significant.
The Friedman test was used to conduct repeated-measures analysis. For multiple comparisons, Bonferroni correction was employed for the repeated-measures analysis. When comparing the left and right eyes of the young and old monkey groups, no significant differences were observed. To simplify the analysis, only the right eye data from the monkey group were included in the subsequent analysis. To minimize type I error, repeated-measures analysis was conducted based on both sex and eye group.
In total, 158 young cynomolgus monkeys aged 3.95 (1.61; range, 1.38-5.97) years and 252 old cynomolgus monkeys aged 18.80 (1.67; range, 15.21-24.69) years were included. Table 1 presents the IOP data stratified by eyes and sex within the young and old monkey groups. No significant differences were observed between different sexes and different eyes.
Table 2 shows the IOP data of the old and young monkey groups. Between-group comparisons and repeated-measures analyses were also performed. The repeated-measures analysis revealed statistically significant differences in IOP between time points (p < 0.001) and interaction effects between time points and groups (p < 0.001). In addition, all pairwise comparisons in the repeated-measures analysis after controlling for significance showed statistically significant differences. IOP was greater in old monkeys than in young monkeys at each time point (p < 0.001), and IOP decreased in three measurements (after anesthesia, before mydriasis, and after mydriasis; p < 0.001).
Figure 2 shows the trend of IOP changes in different periods within both groups of old and young monkeys. The magnitude of the IOP decrease (from after anesthesia to before mydriasis and from before mydriasis to after mydriasis) varied between the age groups. This variation is reflected in the interaction effects observed in the repeated-measures analyses, as well as the different slopes of the graph. After mydriasis, the reduction in the young group was 1.33 (3.67) compared to before mydriasis, while the old monkey group exhibited a decrease of 2.52 (3.94) following dilation. These findings revealed a significant difference between the two groups (p < 0.001).
Regarding sex comparison, no statistically significant difference was observed between male and female monkeys in terms of IOP measured at different time points in both old and young monkey groups. Therefore, no sex bias was used to assess the IOP range at different time points in the entire old or young monkey group. In addition, we conducted further stratified analysis (Appendix 1, hierarchical analysis) and still found no statistical difference between sex and eyes. Table 1 also displays the distribution of IOP based on different sexes to provide additional information.
Figure 3 illustrates the linear and polynomial fitting of IOP distribution in M. fascicularis across different age groups. For the IOP after anesthesia, the adjusted R-squared value for linear fitting was 0.099 (F = 47.76, p < 0.001), while the adjusted R-squared value for polynomial fitting was 0.125 (F = 31.50, p < 0.001). For the IOP before mydriasis, the adjusted R-squared value for linear fitting was 0.279 (F = 165.47, p < 0.001), and the adjusted R-squared value for polynomial fitting was 0.342 (F = 111.94, p < 0.001). For the IOP after mydriasis, the adjusted R-squared value for linear fitting was 0.162 (F = 82.69, p < 0.001), while the adjusted R-squared value for polynomial fitting was 0.201 (F = 54.43, p < 0.001).
Compared to previous studies in cynomolgus monkeys, this study had a larger sample size and standardized the monkey groups into two age groups. This provides a valuable reference for future studies on IOP using the M. fascicularis model. The present study explores the distribution of IOP in different age groups. Additionally, the imposition of an age limit on the monkey groups within this study mitigated the potential impact of diverse systemic factors associated with differing age brackets, thereby reducing the level of confounding variables and enhancing the degree of representativeness. Appendix 2 also shows some other studies on IOP after anesthesia in different animals.
We observed a decreasing trend in IOP from the onset of anesthesia to a certain duration afterward. This trend is consistent with the findings of Jasien et al. [9] in rhesus macaques undergoing general anesthesia. However, human studies have shown that, under initial general anesthesia, IOP shows no statistically significant changes compared to the baseline levels [10]. A large sample study on rhesus monkeys also found no statistically significant difference in IOP between 13 and 104 min of anesthesia with ketamine [11]. Furthermore, the result on trained monkeys shows no statistical difference between before and after anesthesia [11]. However, Hahnenberger’s [12] study reported that IOP decreased after anesthesia, but their sample size was too small (only four cases) and exhibited substantial individual variability. In addition, Bunch et al. [13] found that IOP decreased after anesthesia, but their research on the long-term effect of anesthesia (3-5 days) significantly differs from the 2-h anesthesia in this study. It is unconvincing, therefore, to attribute the decline in IOP we observed after anesthesia and after mydriasis solely to the “anesthesia-induced IOP decline” effect.
Therefore, the decrease in IOP after anesthesia and before mydriasis may be attributed to the “white coat effect” observed in animal experiments [9], which induces stress and leads to elevated IOP even in behaviorally trained mice [14]. In summary, nonhuman primates experience a rapid increase in IOP during periods of stress [8]. For example, stable animals experience an increase in IOP, which is effectively blocked by anesthesia [15]. As the anesthesia takes effect and reaches a stable phase, the effect of tension in the extraocular muscles caused by the experimenter’s activity diminishes, and IOP returns to baseline levels. In the present study, the second measurement was taken after 29 ± 17 min, entering the stable anesthesia period, which we considered closer to the average level in the normal state.
As shown in Figure 2, a clear distinction in the decreasing trend between old and young monkeys was observed after a period of effective anesthesia, as compared to the beginning of anesthesia. In different age groups, the reduction of IOP after anesthesia varies, manifesting in distinct slopes on the graph, which may be attributed to varying degrees of response to the “white coat effect.” In our research, we noted a reduction in IOP after mydriasis in both old and young monkeys, compared to their levels before mydriasis. The impact of mydriasis on IOP in healthy individuals remains contentious, with studies reporting varied outcomes, including increases [5], decreases [14,16], or no significant changes [17] in IOP levels. Many also indicate a considerable individual variation in the changes of IOP following mydriasis [18,19]. The increase of anterior chamber depth and the reduced muscle tone following ciliary muscle paralysis may result in lowered IOP after mydriasis [14,20]. These findings could potentially explain the lower IOP observed in the third measurement compared to the second. Although our study on cynomolgus monkeys revealed a statistically significant decrease in IOP following mydriasis, the clinical significance of this reduction depends on specific circumstances. Further research is required to deepen the understanding of the mechanistic aspects of IOP changes following mydriasis.
Our analysis indicated that the polynomial fitting of age with IOP in M. fascicularis outperformed the linear fitting, suggesting a nonlinear relationship between IOP and age, consistent with findings in humans [2,21-24]. Notably, restricted cubic splines analysis further confirmed the presence of this nonlinear relationship (p < 0.001). To minimize confounding effects due to nonlinear relationships, we chose to analyze the younger and older monkey groups separately. This approach can provide valuable insights for research on congenital and age-related ocular diseases in monkeys.
An additional limitation of this study was the absence of middle-aged monkeys. However, this limitation has a minimal effect on the study’s main objective of investigating the range of IOP in young and old cynomolgus monkeys during stable anesthesia. In the subsequent phase, comprehensive IOP studies across monkey groups of varying ages are required further to investigate the nonlinear relationship between IOP and age. Additionally, individual sensitivity to anesthesia may vary, suggesting the need for additional research.
In old cynomolgus monkeys, IOP was consistently higher compared to that of young cynomolgus monkeys, regardless of whether it was measured after anesthesia, before mydriasis, or after mydriasis. Following anesthesia, the reduction in IOP was greater in young cynomolgus monkeys than in old ones. At the same time, the IOP observed in younger cynomolgus monkeys had less reduction compared to older ones following mydriasis. Furthermore, in the M. fascicularis colony, IOP can be considered equal between males and females or between right and left eyes.
Ethics approval and consent to participate: The statement of Association for Research in Vision and Ophthalmology on the use of animals in ophthalmic and vision research was followed. All animals were handled under protocols approved and regulated by the Ethical Committee of the Guangzhou Huazhen Biosciences Company (Ethics Number: 2020–168) and Zhongshan Ophthalmic Center (Permit Number: SYXK (YUE) 2018–0189). Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by the National Key R&D Project of China (2020YFA0112701); the National Natural Science Foundation of China (82,171,057); Science and Technology Program of Guangzhou, China (202,206,080,005); Major Science and Technology Project of Zhongshan City (2022A1007); Natural Science Foundation of Guangdong Province (2024A1515013058). Authors' contributions: JAZ and ZDL complete the data analysis and manuscript writing, who were major contributors in writing the manuscript. GTY, YZ, JW, WL and KZC collected the data. RX and YTZ polished up the manuscript. YTZ and YHZ provide the idea and censored the whole manuscript finally. All authors read approved the final manuscript.